Detection of small nucleic acids

ABSTRACT

The present invention relates to compositions and methods for the detection and characterization of interfering RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs) and other short nucleic acid molecules. More particularly, the present invention relates to methods for the detection and quantitation of interfering RNA expression. The present invention further provides for the detection of variants and types of miRNAs and siRNAs.

The present application is a divisional of application Ser. No.11/929,878 filed Oct. 30, 2007, now abandoned, which is a continuationof application Ser. No. 10/740,256, filed Dec. 18, 2003, now U.S. Pat.No. 7,851,150, issued Dec. 14, 2010, which claims priority to U.S.Provisional Application Ser. No. 60/434,518, filed Dec. 18, 2002, andU.S. Application Ser. No. 60/443,814, filed Jan. 30, 2003, each of whichare herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thedetection and characterization of interfering RNAs such as micro RNAs(miRNAs) and small interfering RNAs (siRNAs) and other short nucleicacid molecules. More particularly, the present invention relates toimproved methods for the detection and quantitation of interfering RNAexpression. The present invention further provides for the detection ofvariants and types of miRNAs and siRNAs.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encodedas short inverted repeats in the genomes of invertebrates andvertebrates (Ambros, (2001) Cell 107, 823-826; Moss (2002) Curr. Biol.12, R138-R140). miRNAs are modulators of target mRNA translation andstability, although most target mRNAs remain to be identified. miRNAssequence-specifically control translation of target mRNAs by binding tosites of antisense complementarity in 3′ untranslated regions (UTRs)(Ambros, supra; Moss, supra; Lagos-Quintana et al., (2001) Science 294,853-858; Lau et al., (2001) Science 294, 858-862; Lee et al., (2001)Science 294, 862-864).

Several miRNAs, such as let-7 RNA, miR-1, miR-34, miR-60, and miR-87,are highly conserved between invertebrates and vertebrates, implicatingthat they may recognize multiple sites and/or multiple targets ofpresumably conserved function (Lagos-Quintana et al., supra; Lau et al.,supra; Lee et al., supra; Pasquinelli et al., (2000) Nature 408:86). Thesmall temporal RNAs (stRNAs) lin-4 and let-7 represent a subclass ofmiRNAs identified by genetic analysis in Caenorhabditis elegans, whichare developmentally regulated and themselves control developmentalprograms, such as timing of neuronal rewiring, Dauer larva formation,vulva formation, and the terminal differentiation of hypodermal cells.

miRNAs are typically excised from 60- to 70-nucleotide foldback RNAprecursor structures, which are sometimes detected at the onset of miRNAprecursor expression (Grishok et al., (2001) Cell 106, 23-34; Hutvagneret al. (2001) Science 93, 834-838; Ketting et al., (2001) Genes Dev. 15,2654-2659) or during expression of very abundant miRNAs (Lagos-Quintanaet al., supra; Lau et al., supra; Lee et al., supra). Generally, onlyone of the strands of the hairpin precursor molecule is excised andaccumulates, presumably because it is protected by associated proteinsfrom RNA degradation. These putative proteins may mediate thetranslational suppression. The miRNA precursor processing reactionrequires Dicer RNase III and Argonaute family members (Grishok et al.,supra; Hutvagner et al., supra; Ketting et al., supra).

In addition to their impact on gene expression, these small RNAs, oftenin the range of 21-22 nucleotides, may find utility in areas oftherapeutics and drug discovery, e.g. as drug targets or aspharmaceutical agents. Thus, in some circumstances, it may be importantto know approximately how much of each miRNA exists in cells. In somecases, it may further be important to compare levels of miRNA indifferent tissue types or before and after application of a stimulus,e.g. a chemical or physical intervention. Because related siRNAs andmiRNAs may be present in low amounts in cells, it is desirable thatmethods of detection be both sensitive and specific. Moreover, forcertain applications, it may be beneficial to identify methods suitablefor high throughput screening, e.g. homogeneous methods, multiplexedmethods, or those suitable to highly parallel automated manipulation andlimited temperature changes.

Although miRNAs play important roles in the regulation of geneexpression, effective techniques for the detection and quantitation ofmiRNA expression are lacking To date, the principal methods used forquantitation of miRNAs are based on gel electrophoresis. The miRNAs aredetected either by Northern blotting or by the presence of radioactiveRNase-resistant duplexes. Northern blotting and chip hybridizationmethods have relatively low analytical sensitivity (Krichevsky et al.2003), so microgram quantities of RNA are needed for analyses; moreover,transfer of small RNAs to filters can introduce problems withreproducibility of quantitation and is not typically amendable tohigh-throughput. Moreover, detection methods based on RNase resistancerequire highly radioactive probes. Further, assays based solely on probehybridization may not provide adequate discrimination between isotypesclosely related in sequence. Alternative approaches involve cloning themiRNAs and then sequencing the inserts. While this approach may besuitable for discriminating single-base differences between closelyrelated miRNA species, it is time consuming and laborious.

Like miRNAs, small interfering RNAs (siRNAs) are small RNA moleculesinvolved in cell defense, e.g. against viral RNA, via a response termedRNA interference (RNAi) (Cullen, B. R., Nature Immunology, 3: 597-599(2002)). One class of siRNAs is produced through the action of the Dicerenzyme and RNA-induced silencing complex (RISC) protein complex as partof the RNAi response to the presence of double stranded RNA in cells(Khvorova, A. et al., Cell 115: 209-216 (2003)). Another class of siRNAsis synthetic and encompasses short duplexes, usually 21-23 nt withcharacteristic dinucleotide overhangs (Elbashir, S. M. et al., EMBO J.20: 6877-6888 (2001)) introduced directly into cells via transfection orexpression from an introduced vector (Paul, C. P. et al., NatureBiotechnology 20: 505-508 (2002), U.S. Patent Application PublicationNo. 2003/0148519A1, herein incorporated by reference in its entirety forall purposes). In some cases, siRNAs appear to persist as definedsequences, making them analogous in function and composition to miRNAs(Elbashir, S. M. et al., supra). What is needed are efficient andaccurate methods of detecting and quantitating miRNA and siRNA levels.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for thedetection and characterization of small nucleic acids, such asinterfering RNAs and other short nucleic acid molecules. Moreparticularly, the present invention relates to improved methods for thedetection and quantitation of interfering RNA expression.

For example, the present invention provides a method, comprising:hybridizing at least one nucleic acid (e.g., that contains sequence thatis not complementary to the interfering RNA) to a interfering RNA targetto generate a detection structure and detecting the detection structure.In some embodiments, the interfering RNA target is an miRNA. In otherembodiments, the interfering RNA target is an siRNA. In someembodiments, the siRNA is double stranded.

In some embodiments, the detection structure comprises an invasivecleavage structure. For example, in some embodiments, the nucleic acidcomprises first and second oligonucleotides configured to form aninvasive cleavage structure in combination with the miRNA. In someembodiments, the nucleic acid comprises a first oligonucleotideconfigured to form an invasive cleavage structure in combination withsaid miRNA. In some embodiments, the first oligonucleotide comprises a5′ portion and a 3′ portion, wherein said 3′ portion is configured tohybridize to said target sequence, and wherein said 5′ portion isconfigured to not hybridize to said target sequence. In some embodimentsemploying a second oligonucleotide, the second oligonucleotide comprisesa 5′ portion and a 3′ portion, wherein said 5′ portion is configured tohybridize to said target sequence, and wherein said 3′ portion isconfigured to not hybridize to said target sequence. In someembodiments, the detecting step comprises use of an INVADER assay.

In some embodiments, the detection structure comprises a circularoligonucleotide hybridized to said small RNA to generate a circulardetection structure. In some embodiments, the detecting step comprisesuse of a rolling circle replication assay.

In some embodiments, the detecting step(s) comprises use of a detectionassay including, but not limited to, sequencing assays, polymerase chainreaction assays, hybridization assays, hybridization assays employing aprobe complementary to a mutation, microarray assays, bead array assays,primer extension assays, enzyme mismatch cleavage assays, branchedhybridization assays, NASBA assays, molecular beacon assays, cyclingprobe assays, ligase chain reaction assays, invasive cleavage structureassays, ARMS assays, and sandwich hybridization assays. In somepreferred embodiments, the detecting step is carried out in cell lysate.

In some embodiments, the methods of the present invention comprisedetecting a second nucleic acid target. In some preferred embodiments,the second nucleic acid target is RNA. In some particularly preferredembodiments, the second nucleic acid target is U6 RNA or GAPDH mRNA.

In some embodiments, the nucleic acid used to form the detectionstructure comprises a template with one or more sites sufficientlycomplementary to the small RNA so as to allow the RNA to hybridize tothe template and be extended in an extension reaction. In someembodiments, the extension reaction is a polymerase chain reactionwherein one or more RNAs are used as primers in the polymerase chainreaction. In some such embodiments, a single type of RNA binds to twolocations on the template to provide the polymerase chain reactionprimers. In other embodiments, two or more RNAs are used as primers. Insuch embodiments, the detection of an amplification product signifiesthe presence of the two or more RNAs in the sample (i.e., an miRNAmultiplex assay). Similar methods may be employed in a ligase chainreaction where the miRNAs are used as the ligated oligonucleotide(s).

In some embodiments, the method comprises detection of a plurality ofmiRNAs. In some such embodiments, the plurality of miRNAs comprisespolymorphisms of the same miRNA. In other embodiments, the plurality ofmiRNAs comprises different miRNAs (e.g., Let-7, miR-1, miR-1d, miR-135,miR-15, miR-16, miR-124a, or miR125b).

The present invention also provides kits for conducting any of the abovemethods. For example, in some embodiments, the present inventionprovides kits comprising a nucleic acid configured for forming adetection structure when hybridized to an RNA target sequence. In someembodiments, the kits are configured to detect an miRNA. In somepreferred embodiments, kits are configured to detect a Let-7, miR-1,miR-135, miR-15, miR-16, miR-1b, miR-124a, or miR125b miRNA. In somepreferred embodiments, kits are configured to co-detect a second RNAtarget with an miRNA target.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic diagrams of INVADER oligonucleotides,probe oligonucleotides and FRET cassettes for detecting two differentalleles (e.g., differing by a single nucleotide) in a single reaction.

FIG. 2 shows an exemplary detection structure utilized in someembodiments of the present invention.

FIG. 3 shows a second exemplary detection structure utilized in someembodiments of the present invention.

FIG. 4 shows a third exemplary detection structure used in someembodiments of the present invention. A=Universal sequence that is addedto the 3′ and 5′ or probes and INVADER oligonucleotides, respectively.From 5′ to 3′, the probe is composed of the 5′-flap, the miRNAcomplementary region, and the DNA universal sequence “A”. The INVADERoligonucleotide from 5′ to 3′, is composed of the DNA universal sequence“A” and an miRNA complementary region. A′=2′-O-methyl universaloligonucleotide that compliments the sequence “A” and is added to kitsas a standard oligonucleotide.

FIGS. 5A-C show exemplary oligonucleotides for use with the presentinvention. Bases of the target miRNA are underlined in lower case. DNAresidues in probe or INVADER oligonucleotides are in regular type. Lowercase type indicates 2′-O-methyl residues.

FIG. 6 shows the results of temperature optimization experiments forlet-7.

FIG. 7 shows the results of temperature optimization experiments forlet-7.

FIGS. 8A and 8B show the results of limit of detection (LOD) experimentsfor let-7.

FIG. 9 shows the results of cross reactivity experiments using let-7miRNA.

FIG. 10 shows the results of LOD experiments for miR-1.

FIG. 11 shows the results of CLEAVASE enzymes IX and XII comparisonsusing let-7 miRNA.

FIG. 12 shows a partial sequence alignment of U6 RNA sequences fromvarious organisms (SEQ ID NOS:107-114).

FIG. 13 shows the results of temperature optimization experiments formir-135.

FIG. 14 shows the results of LOD experiments for mir-135.

FIG. 15 contains a graphical representation of average counts obtainedfor the detection of let-7 in cell lysates.

FIG. 16 shows the results of miRNA and mRNA in cell lysates with andwithout RNAse A treatment.

FIG. 17 shows results of invasive cleavage assays comparing the effectsof including full-length vs. shortened ARRESTOR oligonucleotides.

FIG. 18 shows the results of temperature optimization experiments usingthe assay designs described in FIG. 16.

FIG. 19 shows the results of temperature optimization experiments usingthe 10-mer probe and 12-mer INVADER oligonucleotide designs.

FIG. 20 shows the results of experiments to compare the LODs of twoalternative oligonucleotide designs.

FIG. 21 shows results comparing the effects of substituting 2′-deoxyresidues for some or all of the 2′-O-methyl residues in the probe andINVADER oligonucleotides.

FIG. 22 shows the results of invasive cleavage assays to detectmiR-124a.

FIGS. 23A-E show the results of experiments to detect five differentmiRNA species in total RNA isolated from 20 different tissue types.

FIG. 24 shows results of experiments testing the effect on miRNAdetection of altering probe and oligonucleotide length.

FIG. 25 shows exemplary invasive cleavage oligonucleotide designs fordetection of an siRNA. Lower case residues indicate 2′-O-methyl.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “miRNA” refers to micro RNA. As used herein,the term “miRNA target sequence” refers to a miRNA that is to bedetected (e.g., in the presence of other nucleic acids). In someembodiments, a miRNA target sequence is a variant of a miRNA.

As used herein, the term “RNA detection structure” refers to a structureformed by hybridizing a nucleic acid (e.g., an oligonucleotide) to anRNA target, e.g., an miRNA or siRNA. In some embodiments, the nucleicacid is a single nucleic acid (e.g., a larger nucleic acid with a smallregion (or regions) of homology to the miRNA). In other embodiments, thenucleic acid comprises two nucleic acids (e.g., that hybridize to themiRNA to form a hairpin (e.g., single or double hairpin) structure). Inpreferred embodiments, miRNA detection structures are capable ofdetection using known nucleic acid detection methods, including, but notlimited to, those disclosed herein.

In some embodiments, RNA detection structures are further modifiedfollowing the hybridization step. For example, in some embodiments, thenucleic acid that is hybridized to the RNA provides a template forextension of the RNA by a nucleic acid polymerase. The RNA is hybridizedto the nucleic acid and is then extended using the polymerase. In otherembodiments, the nucleic acid serves as a template for the hybridizationand ligation of additional nucleic acids to the RNA.

The term “siRNAs” refers to short interfering RNAs. In some embodiments,siRNAs comprise a duplex, or double-stranded region, where each strandof the double-stranded region is about 18 to 25 nucleotides long; thedouble-stranded region can be as short as 16, and as long as 29, basepairs long, where the length is determined by the antisense strand.Often siRNAs contain from about two to four unpaired nucleotides at the3′ end of each strand. SiRNAs appear to function as key intermediates intriggering RNA interference in invertebrates and in vertebrates, and intriggering sequence-specific RNA degradation during posttranscriptionalgene silencing in plants. At least one strand of the duplex ordouble-stranded region of a siRNA is substantially homologous to orsubstantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense” strand; thestrand homologous to the target RNA molecule is the “sense” strand andis also complementary to the siRNA antisense strand. One strand of thedouble stranded region need not be the exact length of the oppositestrand’ thus, one strand may have at least one fewer nucleotides thanthe opposite complementary strand, resulting in a “bubble” or at leastone unmatched base in the opposite strand. One strand of thedouble-stranded region need not be exactly complementary to the oppositestrand; thus, the strand, preferably the sense strand, may have at leastone mismatched base pair.

SiRNAs may also contain additional sequences; non-limiting examples ofsuch sequences include linking sequences, or loops, which connect thetwo strands of the duplex region. This form of siRNAs may be referred to“si-like RNA”, “short hairpin siRNA” where the short refers to theduplex region of the siRNA, or “hairpin siRNA”. Additional non-limitingexamples of additional sequences present in siRNAs include stem andother folded structures. The additional sequences may or may not haveknown functions; non-limiting examples of such functions includeincreasing stability of an siRNA molecule, or providing a cellulardestination signal.

As used herein, the terms “subject” and “patient” refer to any organismsincluding plants, microorganisms and animals (e.g., mammals such asdogs, cats, livestock, and humans).

As used herein, the term “INVADER assay reagents” or “invasive cleavageassay reagents” refers to one or more reagents for detecting targetsequences, said reagents comprising oligonucleotides capable of formingan invasive cleavage structure in the presence of the target sequence.In some embodiments, the INVADER assay reagents further comprise anagent for detecting the presence of an invasive cleavage structure(e.g., a cleavage agent). In some embodiments, the oligonucleotidescomprise first and second oligonucleotides, said first oligonucleotidecomprising a 5′ portion complementary to a first region of the targetnucleic acid and said second oligonucleotide comprising a 3′ portion anda 5′ portion, said 5′ portion complementary to a second region of thetarget nucleic acid downstream of and contiguous to the first portion.In some embodiments, the 3′ portion of the second oligonucleotidecomprises a 3′ terminal nucleotide not complementary to the targetnucleic acid. In preferred embodiments, the 3′ portion of the secondoligonucleotide consists of a single nucleotide not complementary to thetarget nucleic acid. In some embodiments, the first and secondoligonucleotides are covalently coupled to one another (e.g., through alinker).

In some embodiments, the INVADER assay reagents further comprise a solidsupport. For example, in some embodiments, the one or moreoligonucleotides of the assay reagents (e.g., first and/or secondoligonucleotide, whether bridging or non-bridging) is attached to thesolid support. In some embodiments, the INVADER assay reagents furthercomprise a buffer solution. In some preferred embodiments, the buffersolution comprises a source of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ions). Individual ingredients (e.g., oligonucleotides, enzymes, buffers,target nucleic acids) that collectively make up INVADER assay reagentsare termed “INVADER assay reagent components.”

In some embodiments, the INVADER assay reagents further comprise a thirdoligonucleotide complementary to a third portion of the target nucleicacid upstream of the first portion of the first target nucleic acid. Inyet other embodiments, the INVADER assay reagents further comprise atarget nucleic acid. In some embodiments, the INVADER assay reagentsfurther comprise a second target nucleic acid. In yet other embodiments,the INVADER assay reagents further comprise a third oligonucleotidecomprising a 5′ portion complementary to a first region of the secondtarget nucleic acid.

In some specific embodiments, the 3′ portion of the thirdoligonucleotide is covalently linked to the second target nucleic acid.In other specific embodiments, the second target nucleic acid furthercomprises a 5′ portion, wherein the 5′ portion of the second targetnucleic acid is the third oligonucleotide. In still other embodiments,the INVADER assay reagents further comprise an ARRESTOR molecule (e.g.,ARRESTOR oligonucleotide).

The inclusion of 2′ O-methylated ARRESTOR oligonucleotides, which arebase-paired fully to each probe's target-specific region and partiallyto its 5′-flap region, sequesters uncleaved probes and preventsX-structure formation in the secondary reaction, as described in Eis etal., Nature Biotechnology, 19:673-676 (2001), herein incorporated byreference in its entirety for all purposes.

In some preferred embodiments, the INVADER assay reagents furthercomprise reagents for detecting a nucleic acid cleavage product. In someembodiments, one or more oligonucleotides in the INVADER assay reagentscomprise a label. In some preferred embodiments, said firstoligonucleotide comprises a label. In other preferred embodiments, saidthird oligonucleotide comprises a label. In particularly preferredembodiments, the reagents comprise a first and/or a thirdoligonucleotide labeled with moieties that produce a fluorescenceresonance energy transfer (FRET) effect.

In some embodiments one or more the INVADER assay reagents may beprovided in a predispensed format (i.e., premeasured for use in a stepof the procedure without re-measurement or re-dispensing). In someembodiments, selected INVADER assay reagent components are mixed andpredispensed together. In preferred embodiments, predispensed assayreagent components are predispensed and are provided in a reactionvessel (including but not limited to a reaction tube or a well, as in,e.g., a microtiter plate). In particularly preferred embodiments,predispensed INVADER assay reagent components are dried down (e.g.,desiccated or lyophilized) in a reaction vessel.

In some embodiments, the INVADER assay reagents are provided as a kit.As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to delivery systemscomprising two or more separate containers that each contains asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

In some embodiments, the present invention provides INVADER assayreagent kits comprising one or more of the components necessary forpracticing the present invention. For example, the present inventionprovides kits for storing or delivering the enzymes and/or the reactioncomponents necessary to practice an INVADER assay. The kit may includeany and all components necessary or desired for assays including, butnot limited to, the reagents themselves, buffers, control reagents(e.g., tissue samples, positive and negative control targetoligonucleotides, etc.), solid supports, labels, written and/orpictorial instructions and product information, inhibitors, labelingand/or detection reagents, package environmental controls (e.g., ice,desiccants, etc.), and the like. In some embodiments, the kits provide asub-set of the required components, wherein it is expected that the userwill supply the remaining components. In some embodiments, the kitscomprise two or more separate containers wherein each container houses asubset of the components to be delivered. For example, a first container(e.g., box) may contain an enzyme (e.g., structure specific cleavageenzyme in a suitable storage buffer and container), while a second boxmay contain oligonucleotides (e.g., INVADER oligonucleotides, probeoligonucleotides, control target oligonucleotides, etc.).

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include, butare not limited to, dyes; radiolabels such as ³²P; binding moieties suchas biotin; haptens such as digoxgenin; luminogenic, phosphorescent orfluorogenic moieties; mass tags; and fluorescent dyes alone or incombination with moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, characteristics of mass or behavior affected by mass (e.g.,MALDI time-of-flight mass spectrometry; fluorescence polarization), andthe like. A label may be a charged moiety (positive or negative charge)or alternatively, may be charge neutral. Labels can include or consistof nucleic acid or protein sequence, so long as the sequence comprisingthe label is detectable.

As used herein, the term “distinct” in reference to signals refers tosignals that can be differentiated one from another, e.g., by spectralproperties such as fluorescence emission wavelength, color, absorbance,mass, size, fluorescence polarization properties, charge, etc., or bycapability of interaction with another moiety, such as with a chemicalreagent, an enzyme, an antibody, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “5′-A-G-T-3′,” is complementary tothe sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids. Either term may also be usedin reference to individual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid strand, in contrast orcomparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “homology” and “homologous” refers to a degree of identity.There may be partial homology or complete homology. A partiallyhomologous sequence is one that is less than 100% identical to anothersequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanineComplementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi and SantaLucia, Biochemistry 36: 10581-94(1997) include more sophisticated computations which take structural andenvironmental, as well as sequence characteristics into account for thecalculation of T_(m).

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The term “wild-type” refers to a gene or a gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified,” “mutant,” or “polymorphic” refers to a gene or gene productthat displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired heterologous sequence. For example, although theterm is not limited to the use of expressed sequences or sequences thatencode an expression product, in some embodiments, the heterologoussequence is a coding sequence and appropriate DNA sequences necessaryfor either the replication of the coding sequence in a host organism, orthe expression of the operably linked coding sequence in a particularhost organism. DNA sequences necessary for expression in prokaryotesinclude a promoter, optionally an operator sequence, a ribosome bindingsite and possibly other sequences. Eukaryotic cells are known to utilizepromoters, polyadenlyation signals and enhancers.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “cleavage structure” as used herein, refers to a structure thatis formed by the interaction of at least one probe oligonucleotide and atarget nucleic acid, forming a structure comprising a duplex, theresulting structure being cleavable by a cleavage means, including butnot limited to an enzyme. The cleavage structure is a substrate forspecific cleavage by the cleavage means in contrast to a nucleic acidmolecule that is a substrate for non-specific cleavage by agents such asphosphodiesterases, which cleave nucleic acid molecules without regardto secondary structure (i.e., no formation of a duplexed structure isrequired).

The term “cleavage means” or “cleavage agent” as used herein refers toany means that is capable of cleaving a cleavage structure, includingbut not limited to enzymes. “Structure-specific nucleases” or“structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

The cleavage means may include nuclease activity provided from a varietyof sources including the CLEAVASE enzymes (Third Wave Technologies,Madison, Wis.), the FEN-1 endonucleases (including RAD2 and XPGproteins), Taq DNA polymerase and E. coli DNA polymerase I. The cleavagemeans may include enzymes having 5′ nuclease activity (e.g., Taq DNApolymerase (DNAP), E. coli DNA polymerase I). The cleavage means mayalso include modified DNA polymerases having 5′ nuclease activity butlacking synthetic activity. Examples of cleavage means suitable for usein the methods and kits of the present invention are provided in U.S.Pat. Nos. 5,614,402; 5,795,763; 5,843,669; PCT Appln. Nos WO 98/23774;WO 02/070755A2; and WO0190337A2, each of which is herein incorporated byreference it their entireties.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher (e.g., including, but not limited to, 60° C., 65° C., 70° C.,75° C., 80° C., 85° C. or 90° C.).

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The term “non-target cleavage product” refers to a product of a cleavagereaction that is not derived from the target nucleic acid. As discussedabove, in some of the methods of the present invention, cleavage of thecleavage structure generally occurs within the probe oligonucleotide.The fragments of the probe oligonucleotide generated by this targetnucleic acid-dependent cleavage are “non-target cleavage products.”

The term “probe oligonucleotide”, in the context of an INVADER assayreaction, refers to an oligonucleotide that interacts with a targetnucleic acid to form a cleavage structure in the presence or absence ofan INVADER oligonucleotide. When annealed to the target nucleic acid,the probe oligonucleotide and target form a cleavage structure andcleavage occurs within the probe oligonucleotide.

The term “INVADER oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid at a location near the region ofhybridization between a probe and the target nucleic acid, wherein theINVADER oligonucleotide comprises a portion (e.g., a chemical moiety, ornucleotide-whether complementary to that target or not) that overlapswith the region of hybridization between the probe and target. In someembodiments, the INVADER oligonucleotide contains sequences at its 3′end that are substantially the same as sequences located at the 5′ endof a probe oligonucleotide.

The term “ARRESTOR molecule” refers to an agent added to or included inan invasive cleavage reaction in order to stop one or more reactioncomponents from participating in a subsequent action or reaction. Thismay be done by sequestering or inactivating some reaction component(e.g., by binding or base-pairing a nucleic acid component, or bybinding to a protein component). The term “ARRESTOR oligonucleotide”refers to an oligonucleotide included in an invasive cleavage reactionin order to stop or arrest one or more aspects of any reaction (e.g.,the first reaction and/or any subsequent reactions or actions; it is notintended that the ARRESTOR oligonucleotide be limited to any particularreaction or reaction step). This may be done by sequestering somereaction component (e.g., base-pairing to another nucleic acid, orbinding to a protein component). However, it is not intended that theterm be so limited as to just situations in which a reaction componentis sequestered.

The term “cassette” as used herein refers to an oligonucleotide orcombination of oligonucleotides configured to generate a detectablesignal in response to cleavage of a probe oligonucleotide in an INVADERassay. In preferred embodiments, the cassette hybridizes to a non-targetcleavage product from cleavage of the probe oligonucleotide to form asecond invasive cleavage structure, such that the cassette can then becleaved.

Secondary cleavage reactions in some preferred embodiments of thepresent invention include the use of FRET cassettes. Such moleculesprovide both a secondary target (Secondary Reaction Target or SRT) and aFRET labeled cleavable sequence, allowing homogeneous detection (i.e.,without product separation or other manipulation after the reaction) ofthe sequential invasive cleavage reaction. Other preferred embodimentsuse a secondary reaction system in which the FRET probe and synthetictarget are provided as separate oligonucleotides. The cleaved 5′-flapsfrom a primary reaction act as invasive oligonucleotides in a secondaryreaction, in which they bind to the appropriate secondary-reactiontarget (SRT).

In some embodiments, the cassette is a single oligonucleotide comprisinga hairpin portion (i.e., a region wherein one portion of the cassetteoligonucleotide hybridizes to a second portion of the sameoligonucleotide under reaction conditions, to form a duplex). In otherembodiments, a cassette comprises at least two oligonucleotidescomprising complementary portions that can form a duplex under reactionconditions. In preferred embodiments, the cassette comprises a label. Inparticularly preferred embodiments, cassette comprises labeled moietiesthat produce a fluorescence resonance energy transfer (FRET) effect.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

As used herein, the phrase “non-amplified oligonucleotide detectionassay” refers to a detection assay configured to detect the presence orabsence of a particular target sequence (e.g., miRNA, SNP, repeatsequence, etc.) that has not been amplified (e.g., by PCR), withoutcreating copies of the target sequence. A “non-amplified oligonucleotidedetection assay” may, for example, amplify a signal used to indicate thepresence or absence of a particular target sequence or polymorphismwithin a target sequence, so long as the target sequence is not copied.

As used herein, the phrase “non-amplifying oligonucleotide detectionassay” refers to a detection assay configured to detect the presence orabsence of a target sequence (e.g., miRNA, SNP, repeat sequence, etc.),without creating copies of the target sequence. A “non-amplifyingoligonucleotide detection assay” may, for example, amplify a signal usedto indicate the presence or absence of a particular target sequence orpolymorphism in a target sequence, so long as the target sequence is notcopied.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides including but not limited to analogsthat have altered stacking interactions such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogenbonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner and herein incorporated by reference); non-hydrogen bondinganalogs (e.g., non-polar, aromatic nucleoside analogs such as2,4-difluorotoluene, described by B. A. Schweitzer and E. T. Kool, J.Org. Chem., 1994, 59, 7238-7242, B. A. Schweitzer and E. T. Kool, J. Am.Chem. Soc., 1995, 117, 1863-1872; each of which is herein incorporatedby reference); “universal” bases such as 5-nitroindole and3-nitropyrrole; and universal purines and pyrimidines (such as “K” and“P” nucleotides, respectively; P. Kong, et al., Nucleic Acids Res.,1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20,5149-5152). Nucleotide analogs include nucleotides having modificationon the sugar moiety, such as dideoxy nucleotides and 2′-O-methylnucleotides. Nucleotide analogs include modified forms ofdeoxyribonucleotides as well as ribonucleotides.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (e.g., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin.

Biological samples may be animal, including human, fluid, solid (e.g.,stool) or tissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,lagomorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

The term “source of target nucleic acid” refers to any sample thatcontains nucleic acids (RNA (e.g., miRNA) or DNA). Particularlypreferred sources of target nucleic acids are biological samplesincluding, but not limited to blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum and semen.

An oligonucleotide is said to be present in “excess” relative to anotheroligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present in at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

A sample “suspected of containing” a first and a second target nucleicacid may contain either, both or neither target nucleic acid molecule.

The term “reactant” is used herein in its broadest sense. The reactantcan comprise, for example, an enzymatic reactant, a chemical reactant orlight (e.g., ultraviolet light, particularly short wavelengthultraviolet light is known to break oligonucleotide chains). Any agentcapable of reacting with an oligonucleotide to either shorten (e.g.,cleave) or elongate the oligonucleotide is encompassed within the term“reactant.”

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, in some embodiments,recombinant CLEAVASE nucleases are expressed in bacterial host cells andthe nucleases are purified by the removal of host cell proteins; thepercent of these recombinant nucleases is thereby increased in thesample.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid (e.g., 4, 5, 6, . . . , n−1).

The term “nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin, which may besingle or double stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence.

As used herein, the terms “purified” or “substantially purified” referto molecules, either nucleic or amino acid sequences, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

The term “continuous strand of nucleic acid” as used herein is means astrand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

The term “continuous duplex” as used herein refers to a region of doublestranded nucleic acid in which there is no disruption in the progressionof basepairs within the duplex (i.e., the base pairs along the duplexare not distorted to accommodate a gap, bulge or mismatch with theconfines of the region of continuous duplex). As used herein the termrefers only to the arrangement of the basepairs within the duplex,without implication of continuity in the backbone portion of the nucleicacid strand. Duplex nucleic acids with uninterrupted basepairing, butwith nicks in one or both strands are within the definition of acontinuous duplex.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for thedetection and characterization of micro RNAs (miRNAs) or other shortnucleic acid molecules, e.g. siRNAs. The present invention providesimproved methods of detecting, characterizing and quantitating miRNAexpression. In some embodiments, the present invention provides methodsof detecting miRNA expression comprising adding a nucleic acid to amiRNA to aid in detection. The resulting “miRNA detection structure” isthen detected using any suitable method including, but not limited to,those disclosed herein. While the following description focuses on thedetection and quantitation of miRNAs, it should be understood that theinvention also finds use with other short nucleic acid molecules (e.g.,DNA and RNA of less than, for example, 50, 40, 30, or 20 nucleotides inlength).

I. Formation of a miRNA Detection Structure

In some embodiments, the present invention provides methods ofgenerating miRNA detection structures to aid in the detection of miRNAs.miRNAs are small in size (approximately 21 nucleotides) and are thusdifficult to detect using standardized hybridization methods. In someembodiments, the methods of the present invention comprise adding anucleic acid molecule to an miRNA (e.g., via hybridization, extension,or ligation) to generate a detection structure. Such detectionstructures can then be detected using any suitable method.

In one particular embodiment, the detection structure described in FIG.2 is generated for detection of miRNAs. In this embodiment, twooligonucleotides are annealed to the miRNA to form a double loop or“dumbbell” like structure. The dumbbell structure creates a largerregion of double-stranded nucleic acid by extending the ends of themiRNA with a double-stranded region of oligonucleotide. In someembodiments, each end of the miRNA is extended between 2 and 5nucleotides. In some embodiments, the ends of the oligonucleotidescomprise additional nucleic acid sequences that do not hybridize to themiRNA. In some embodiments, these additional sequences form invasivecleavage structures (e.g., INVADER assay invasive cleavage structures).In some embodiments, invasive cleavage structures are detected by theINVADER assay (See e.g., below description).

In other embodiments, the detection structure described in FIG. 3 isgenerated for the detection of miRNAs. In this embodiment, oneoligonucleotide is annealed to the miRNA to generate an archedstructure. The miRNA brings the ends of the oligonucleotide togetherwith greater efficiency than in the absence of the miRNA. In someembodiments, the ends of the oligonucleotide comprise additionalsequences that extend beyond the miRNA and do not hybridize to themiRNA. In some embodiments, these additional sequences form invasivecleavage structures (e.g., INVADER assay invasive cleavage structures).In some embodiments, invasive cleavage structures are detected by theINVADER assay (See e.g., below description). In other embodiments,following cleavage of an INVADER assay invasive cleavage structure, theresulting ends are ligated to form a circular structure. In otherembodiments, one oligonucleotide is hybridized to a miRNA such that theends of the oligonucleotide are brought in close proximity (e.g.,hybridized to adjacent nucleotides of the miRNA) and are then ligated.

In still further embodiments, the detection structures described inFIGS. 16 and 17 are generated. In this embodiment, either probe orINVADER oligonucleotides are extended to create a single hairpin loop or“half dumbbell” structure. In some embodiments, the ends of theoligonucleotides comprise additional nucleic acid sequences that do nothybridize to the miRNA. In some embodiments, these additional sequencesform invasive cleavage structures (e.g., INVADER assay invasive cleavagestructures). In some embodiments, invasive cleavage structures aredetected by the INVADER assay (See e.g., below description).

In other embodiments, these additional sequences are complementary toadditional oligonucleotides added to reaction mixtures to stabilize acleavage structure, e.g. an INVADER assay invasive cleavage structure(FIG. 4).

In some embodiments, circular structures generated as described aboveare detected using a rolling circle replication assay (See e.g., belowdescription of rolling circle replication).

In still further embodiments, detection structures are generated fromlong oligonucleotides (e.g., greater than 50, 100, 1000 or morenucleotides) with short region(s) of homology to miRNAs. One or moremiRNAs are hybridized to the oligonucleotides to generate detectionstructures. In some embodiments, these detection structures are detectedby extension of miRNAs (e.g., via ligation or polymerization reactionssuch as RT-PCR). In some embodiments, these detection structures arefurther detected by hybridization to oligonucleotides conjugated tosolid supports, such as microspheres, or other surfaces or structures.

In some embodiments, oligonucleotides used to form detection structurescomprise one or more nucleotide analogs. For example, in someembodiments, 2′-O-methyl nucleotides are utilized. The present inventionis not limited to a particular mechanism. Indeed, an understanding ofthe mechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that the presence of 2′-O-methyl basesincreases the stability of the hybridized detection structure and aidsin further detection methods.

II. Detection of Interfering RNAs

In some embodiments, the present invention provides methods of detectingmiRNAs. The present invention is not limited to a particular detectionassay. Any suitable method may be utilized including, but not limitedto, those disclosed herein.

In some preferred embodiments of the present invention, miRNA detectionmethods are quantitative. The present invention is not limited to aparticular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, it iscontemplated that levels of a particular miRNA in the body areassociated with a level of gene expression from their cognate genes. Thepresent invention thus provides methods of correlated miRNAs with geneexpression of particular genes (e.g., genes involved in disease statesor metabolism). For example, in some embodiments, the methods of thepresent invention are utilized to determine the presence of abnormal(e.g., high or low) levels of a particular miRNA or to determine theeffect of an intervention (e.g., drug) on miRNA expression. In otherembodiments, heterologous miRNAs (e.g., from expression vectors,transgenic constructs, transfection, etc.) are detected to characterizethe efficiency of miRNA expression systems.

In some embodiments, the present invention provides methods of detectinga particular miRNA (e.g., a miRNA such as mir-1 or mir-135). In otherembodiments, the methods of the present invention are used todistinguish between variants (e.g., polymorphisms or mutations) in aparticular miRNA. In still further embodiments, the present inventionprovides methods of lysing cells to be tested for the presence ofmiRNAs.

A. INVADER Assay

In some embodiments, the INVADER assay is used for the detection ofmiRNAs. In some embodiments, the INVADER assay comprises forming anucleic acid cleavage structure that is dependent upon the presence of atarget nucleic acid and cleaving the nucleic acid cleavage structure soas to release distinctive cleavage products. 5′ nuclease activity, forexample, is used to cleave the target-dependent cleavage structure andthe resulting cleavage products or the cleavage of the cleavagestructure is indicative of the presence of specific target nucleic acidsequences in the sample. When one or two (or more) strands of nucleicacid, or oligonucleotides, both hybridize to a target nucleic acidstrand such that they form an overlapping invasive cleavage structure,as described below, invasive cleavage can occur. Through the interactionof a cleavage agent (e.g., a 5′ nuclease) and the upstreamoligonucleotide, the cleavage agent can be made to cleave the downstreamoligonucleotide at an internal site in such a way that a distinctivefragment is produced. Such embodiments have been termed the INVADERassay (Third Wave Technologies) and are described in U.S. Pat. Nos.5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, 6,348,314, and6,458,535, WO 97/27214 WO 98/42873, Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), each of which isherein incorporated by reference in its entirety for all purposes).

The INVADER assay detects hybridization of probes to a target byenzymatic cleavage of specific structures by structure specific enzymes(See, INVADER assays, Third Wave Technologies; See e.g., U.S. Pat. Nos.5,846,717; 6,090,543; 6,001,567; 5,985,557; 5,994,069; 6,090,543;6,348,314; 6,458,535; U.S. Pat. App. Nos. 20030186238 (Ser. No.10/084,839); 20030104378A1 (Ser. No. 09/864,636); Lyamichev et al., Nat.Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000),WO97/27214 and WO98/42873, each of which is herein incorporated byreference in its entirety for all purposes).

The INVADER assay detects specific DNA and RNA sequences by usingstructure-specific enzymes (e.g. FEN endonucleases) to cleave a complexformed by the hybridization of overlapping oligonucleotide probes (See,e.g. FIG. 1). Elevated temperature and an excess of one of the probesenable multiple probes to be cleaved for each target sequence presentwithout temperature cycling. In some embodiments, these cleaved probesthen direct cleavage of a second labeled probe. The secondary probeoligonucleotide can be 5′-end labeled with fluorescein that is quenchedby an internal dye. Upon cleavage, the de-quenched fluorescein labeledproduct may be detected using a standard fluorescence plate reader.

The INVADER assay detects specific sequences, mutations, and SNPs inunamplified, as well as amplified, RNA and DNA, including genomic DNA.In the embodiments shown schematically in FIG. 1, the INVADER assay usestwo cascading steps (a primary and a secondary reaction) both togenerate and then to amplify the target-specific signal. Forconvenience, the alleles in the following discussion are described aswild-type (WT) and mutant (MT), even though this terminology does notapply to all genetic variations. In the primary reaction (FIG. 1, panelA), the WT primary probe and the INVADER oligonucleotide hybridize intandem to the target nucleic acid to form an overlapping structure. Anunpaired “flap” is included on the 5′ end of the WT primary probe. Astructure-specific enzyme (e.g. the CLEAVASE enzyme, Third WaveTechnologies) recognizes the overlap and cleaves off the unpaired flap,releasing it as a target-specific product. In the secondary reaction,this cleaved product serves as an INVADER oligonucleotide on the WTfluorescence resonance energy transfer (WT-FRET) probe to again createthe structure recognized by the structure specific enzyme (panel A).When the two dyes on a single FRET probe are separated by cleavage(indicated by the arrow in FIG. 1), a detectable fluorescent signalabove background fluorescence is produced. Consequently, cleavage ofthis second structure results in an increase in fluorescence, indicatingthe presence of the WT allele (or mutant allele if the assay isconfigured for the mutant allele to generate the detectable signal). Insome embodiments, FRET probes having different labels (e.g. resolvableby difference in emission or excitation wavelengths, or resolvable bytime-resolved fluorescence detection) are provided for each allele orlocus to be detected, such that the different alleles or loci can bedetected in a single reaction. In such embodiments, the primary probesets and the different FRET probes may be combined in a single assay,allowing comparison of the signals from each allele or locus in the samesample.

If the primary probe oligonucleotide and the target nucleotide sequencedo not match perfectly at the cleavage site (e.g., as with the MTprimary probe and the WT target, FIG. 1, panel B), the overlappedstructure does not form and cleavage is suppressed. The structurespecific enzyme (e.g., CLEAVASE VIII enzyme, Third Wave Technologies)used cleaves the overlapped structure more efficiently (e.g. at least340-fold) than the non-overlapping structure, allowing excellentdiscrimination of the alleles. The probes turn over without temperaturecycling to produce many signals per target (i.e., linear signalamplification). Similarly, each target-specific product can enable thecleavage of many FRET probes.

The primary INVADER assay reaction is directed against the target DNA(or RNA) being detected. The target DNA is the limiting component in thefirst invasive cleavage, since the INVADER and primary probe aresupplied in molar excess. In the second invasive cleavage, it is thereleased flap that is limiting. When these two cleavage reactions areperformed sequentially, the fluorescence signal from the compositereaction accumulates linearly with respect to the target DNA amount.

In certain embodiments, the INVADER assay, or other nucleotide detectionassays, are performed with accessible site-designed oligonucleotidesand/or bridging oligonucleotides. Such methods, procedures andcompositions are described in U.S. Pat. Nos. 6,194,149, 6,358,691, 6355,437, U.S. patent application Ser. No. 09/882,945, and PCT ApplicationsWO9850403, and WO0198537, all of which are specifically incorporated byreference in their entireties.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

In some embodiments, the target sequence comprises a first region and asecond region, the second region downstream of and contiguous to thefirst region, and the oligonucleotides comprise first and secondoligonucleotides, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thetarget sequence and wherein the second oligonucleotide comprises a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of the target nucleic acid.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between the target sequence and theoligonucleotides if the target sequence is present in the sample,wherein the invasive cleavage structure is cleaved by the cleavage agentto form a cleavage product.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

In some particularly preferred embodiments, the target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and saidoligonucleotides comprise first and second oligonucleotides, wherein atleast a portion of said first oligonucleotide is completelycomplementary to said first portion of said target sequence and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said target nucleic acid.

In certain embodiments, the present invention provides kits for assayinga pooled sample (e.g., a pooled blood sample or pooled cell lysates)using INVADER detection reagents (e.g. primary probe, INVADER probe, andFRET cassette). In preferred embodiments, the kit further comprisesinstructions on how to perform the INVADER assay, and in someembodiments, how to apply the INVADER detection assay to pooled samplesfrom many individuals, or to “pooled” samples from many cells (e.g.,from a biopsy sample) from a single subject.

The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.g.through probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, (Reynaldo et al., J. Mol. Biol. 97: 511-520(2000)), multiple probes can hybridize to the same target, allowingmultiple cleavages, and the generation of multiple cleavage products.

The INVADER Assay Reaction:

In preferred embodiments of the INVADER DNA assay, two oligonucleotides(a discriminatory Primary Probe and an INVADER Oligo) hybridize intandem to the target DNA to form an overlapping structure. The 5′-end ofthe Primary Probe includes a 5′-flap that does not hybridize to thetarget DNA (FIG. 1). The 3′-nucleotide of the bound INVADERoligonucleotide overlaps the Primary Probe, but need not hybridize tothe target DNA. The CLEAVASE enzyme recognizes this overlappingstructure and cleaves off the unpaired 5′-flap of the Primary Probe,releasing it as a target-specific product. The Primary Probe is designedto have a melting temperature close to the reaction temperature. Thus,under the isothermal assay conditions, Primary Probes, which areprovided in excess, cycle on the target DNA. This allows for multiplerounds of Primary Probe cleavage for each target DNA, and amplificationof the number of released 5′-flaps.

In the secondary reaction, each released 5′-flap can serve as an INVADERoligonucleotide on a fluorescence resonance energy transfer (FRET)Cassette to create another overlapping structure that is recognized andcleaved by the CLEAVASE enzyme (FIG. 1). When the FRET Cassette iscleaved, the fluorophore (F) and quencher (Q) are separated, generatingdetectable fluorescence signal. Similar to the initial reaction, thereleased 5′-flap and the FRET Cassette cycle, resulting in amplifiedfluorescence signal. The initial and secondary reactions runconcurrently in the same well.

The biplex format of the INVADER DNA Assay enables simultaneousdetection of two DNA sequences in a single well. Most often, thisinvolves detection of two variants of a particular polymorphism (e.g.,in a miRNA). The biplex format uses two different discriminatory PrimaryProbes, each with a unique 5′-flap, and two different FRET Cassettes,each with a spectrally distinct fluorophore. By design, the released5′-flaps will bind only to their respective FRET Cassettes to generate atarget-specific signal.

In some embodiments, the present invention provides kits comprising oneor more of the components necessary for practicing the presentinvention. For example, the present invention provides kits for storingor delivering the enzymes of the present invention and/or the reactioncomponents necessary to practice a cleavage assay (e.g., the INVADERassay). By way of example, and not intending to limit the kits of thepresent invention to any particular configuration or combination ofcomponents, the following section describes one embodiment of a kit forpracticing the present invention:

In some embodiments, the kits of the present invention provide thefollowing reagents:

CLEAVASE enzyme Primary Probe Oligos DNA Reaction Buffer 1 INVADER OligoFRET Cassette 1 (e.g., F) FRET Cassette 2 (e.g., R) Mutant DNA controlsWild type DNA controls “No Target” Blank controlIn other embodiments, the kits of the present invention are configuredfor direct detection of RNA. These kits may provide the followingreagents:

CLEAVASE enzyme Primary Probe oligonucleotides DNA Reaction Buffer 1INVADER Oligo FRET Probe 1 (e.g., F) FRET Probe 2 (e.g., R) SecondaryReaction Target 1 Secondary Reaction Target 2 ARRESTOR oligonucleotide 1ARRESTOR oligonucleotide 2 Mutant DNA controls Wild type DNA controls“No Target” Blank control

An additional consideration has to do with undesired effects resultingfrom particular combinations of oligonucleotides in a single reaction.One such effect is target-independent generation of background signal.Certain oligonucleotides in combination with others may generate signalin the INVADER assay in the absence of the particular target beingdetected. Separation of these oligonucleotide combinations intodifferent pools can be used to alleviate this effect. Similarly, certainoligonucleotide combinations can artificially repress signal generationfrom a desired target. Again, separation of these combinations intodifferent pools can alleviate this effect.

The designs of the probes sets (e.g., the oligonucleotides and/or theirsequences) are adapted for use in miRNA detection assays using theguidelines for reaction design and optimization provided herein (Seee.g., the Experimental Section). For example, in some embodiments, thereaction temperature is reduced (e.g., to 50-60° C.) to account for thesmaller region of hybridization.

In some embodiments, a kit of the present invention provides a list ofadditional components (e.g., reagents, supplies, and/or equipment) to besupplied by a user in order to perform the methods of the invention. Forexample, and without intending to limit such additional components liststo any particular components, one embodiment of such a list comprisesthe following:

-   -   Clear CHILLOUT-14 liquid wax (MJ Research) or RNase-free,        optical grade mineral oil (Sigma, Cat. No. M-5904)    -   96-well polypropylene microplate (MJ Research, Cat. No.        MSP-9601)    -   Sterile 1.5-ml or 2.0-ml microcentrifuge tubes    -   Sterile, DNase/RNase free disposable aerosol barrier pipet tips    -   Multichannel pipets (0.5-10 μl, 2.5-20 μl)    -   Thermal cycler or other heat source (e.g., lab oven or heating        block).    -   Miscellaneous laboratory equipment (tube racks, micropipetors,        multichannel pipet, microcentrifuge, vortex mixer).    -   Fluorescence microplate reader (a preferred plate reader is        top-reading and equipped with light filters have the following        characteristics:

Excitation Emission (Wavelength/Bandwidth) (Wavelength/Bandwidth) 485nm/20 nm 530 nm/25 nm 560 nm/20 nm 620 nm/40 nm

In some embodiments, a kit of the present invention provides a list ofoptional components (e.g., reagents, supplies, and/or equipment) to besupplied by a user to facilitate performance of the methods of theinvention. For example, and without intending to limit such optionalcomponents lists to any particular components, one embodiment of such alist comprises the following:

Sterile 8-tube strip or microplate (optional)

Disposable plastic trough (optional)

Plate sealing tape (optional)

In some embodiments, a kit of the present invention provides a list ofrequired components to be supplied by a user to facilitate performanceof the methods of the invention for which multiple alternatives areacceptable (e.g. sample preparation kits). For example, and withoutintending to limit such optional components lists to any particularcomponents, one embodiment of such a list comprises the following:

QIAGEN QIAAMP Blood Kit

Gentra Systems PUREGENE Kit

Gentra Systems GENERATION Products

In some embodiments of a kit, detailed protocols are provided. Inpreferred embodiments, protocols for the assembly of INVADER assayreactions (e.g., formulations and preferred procedures for makingreaction mixtures) are provided. In particularly preferred embodiments,protocols for assembly of reaction mixtures include computational orgraphical aids to reduce risk of error in the performance of the methodsof the present invention (e.g., tables to facilitate calculation ofvolumes of reagents needed for multiple reactions, and plate-layoutguides to assist in configuring multi-well assay plates to containnumerous assay reactions).

In some embodiments, supplementary documentation, such as protocols forancillary procedures, e.g., for the preparation of additional reagents,or for preparation of samples for use in the methods of the presentinvention, are provided. In preferred embodiments, supplementarydocumentation includes guidelines and lists of precautions provided tofacilitate successful use of the methods and kits by unskilled orinexperienced users. In particularly preferred embodiments,supplementary documentation includes a troubleshooting guide, e.g., aguide describing possible problems that may be encountered by users, andproviding suggested solutions or corrections to intended to aid the userin resolving or avoiding such problems.

In preferred embodiments, samples are diluted to concentrations thatcorrespond to a 10-μl addition per reaction. The concentration of a100-ng sample should be 15 ng/μl.

B. Rolling Circle Replication

In other embodiments, rolling circle replication methods (AmershamBiosciences, Piscataway, N.J.) are utilized for detection of miRNAdetection structures (See e.g., U.S. Pat. Nos. 6,344,329; 6,143,495;6,316,229; 6,210,884, 6,183,960 and 6,235,502; each of which is hereinincorporated by reference). In some embodiments, rolling circlereplication is used to detect circular miRNA detection structuresgenerated from the annealing of the ends of a single oligonucleotideannealed to a miRNA. In some embodiments, the ends of theoligonucleotide hybridize to the miRNA with no overlap. Thisoligonucleotide can be ligated in the presence or absence of miRNA.However, the ligation reaction is more efficient in the presence of themiRNA. In such embodiments, the level of circular molecules detectedover time is compared to a control reaction lacking miRNA.

In other embodiments, the ends of the oligonucleotide hybridize to themiRNA with overlapping ends to generate an invasive cleavage structure.Such structures are cleaved prior to ligation, thus improving thespecificity of the generation of the circular detection structure.

Rolling circle amplification (RCA) involves replication of circularsingle-stranded DNA molecules. In RCA, a rolling circle replicationprimer hybridizes to circular nucleic acid molecules followed by rollingcircle replication of the nucleic acid molecules using astrand-displacing DNA polymerase. Amplification takes place duringrolling circle replication in a single reaction cycle. Rolling circlereplication results in large DNA molecules containing tandem repeats ofthe nucleic acid sequence. This DNA molecule is referred to as a tandemsequence DNA (TS-DNA).

In some embodiments, ligation-mediated rolling circle amplification(LM-RCA), which involves a ligation operation prior to replication, isutilized. In the ligation operation, an probe hybridizes to its cognatetarget nucleic acid sequence, if present, followed by ligation of theends of the hybridized probe to form a covalently closed,single-stranded nucleic acid. After ligation, a rolling circlereplication primer hybridizes to probe molecules followed by rollingcircle replication of the circular molecules using a strand-displacingDNA polymerase. Generally, LM-RCA comprises mixing an open circle probewith a target sample, resulting in an probe-target sample mixture, andincubating the probe-target sample mixture under conditions promotinghybridization between the open circle probe and a target sequence,mixing ligase with the probe-target sample mixture, resulting in aligation mixture, and incubating the ligation mixture under conditionspromoting ligation of the open circle probe to form an amplificationtarget circle (ATC), mixing a rolling circle replication primer (RCRP)with the ligation mixture, resulting in a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circle and the rollingcircle replication primer, mixing DNA polymerase with the primer-ATCmixture, resulting in a polymerase-ATC mixture, and incubating thepolymerase-ATC mixture under conditions promoting replication of theamplification target circle, where replication of the amplificationtarget circle results in formation of tandem sequence DNA (TS-DNA).

C. Additional Detection Methods

The present invention is not limited to INVADER assay or rolling circleassay detection. Any method that allows for the detection of miRNAdetection structures may be utilized. Exemplary, non-limiting detectionassay that find use in the methods of the present invention aredescribed below.

1. Hybridization Assays

In some embodiments of the present invention, detection structures aredetected using a hybridization assay. In a hybridization assay, thepresence of absence of a given nucleic acid sequence is determined basedon the ability of the DNA from the sample to hybridize to acomplementary DNA molecule (e.g., a oligonucleotide probe). A variety ofhybridization assays using a variety of technologies for hybridizationand detection are available. A description of a selection of assays isprovided below.

a. Direct Detection of Hybridization

In some embodiments, hybridization of a probe to the sequence ofinterest (e.g., a SNP or mutation) is detected directly by visualizing abound probe (e.g., a Northern or Southern assay; See e.g., Ausabel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. [1991]). In a these assays, genomic DNA (Southern) or RNA(Northern) is isolated from a subject. The DNA or RNA is then cleavedwith a series of restriction enzymes that cleave infrequently in thegenome and not near any of the markers being assayed. The DNA or RNA isthen separated (e.g., on an agarose gel) and transferred to a membrane.A labeled (e.g., by incorporating a radionucleotide) probe or probesspecific for the SNP or mutation being detected is allowed to contactthe membrane under a condition of low, medium, or high stringencyconditions. Unbound probe is removed and the presence of binding isdetected by visualizing the labeled probe.

b. Detection of Hybridization Using “DNA Chip” Assays

In some embodiments of the present invention, variant sequences aredetected using a DNA chip hybridization assay. In this assay, a seriesof oligonucleotide probes are affixed to a solid support. Theoligonucleotide probes are designed to be unique to a given targetsequence (e.g., miRNA target sequence). The DNA sample of interest iscontacted with the DNA “chip” and hybridization is detected.

In some embodiments, the DNA chip assay is a GeneChip (Affymetrix, SantaClara, Calif.; See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; and5,858,659; each of which is herein incorporated by reference) assay. TheGeneChip technology uses miniaturized, high-density arrays ofoligonucleotide probes affixed to a “chip.” Probe arrays aremanufactured by Affymetrix's light-directed chemical synthesis process,which combines solid-phase chemical synthesis with photolithographicfabrication techniques employed in the semiconductor industry. Using aseries of photolithographic masks to define chip exposure sites,followed by specific chemical synthesis steps, the process constructshigh-density arrays of oligonucleotides, with each probe in a predefinedposition in the array. Multiple probe arrays are synthesizedsimultaneously on a large glass wafer. The wafers are then diced, andindividual probe arrays are packaged in injection-molded plasticcartridges, which protect them from the environment and serve aschambers for hybridization.

The nucleic acid to be analyzed is isolated, amplified by PCR, andlabeled with a fluorescent reporter group. The labeled DNA is thenincubated with the array using a fluidics station. The array is theninserted into the scanner, where patterns of hybridization are detected.The hybridization data are collected as light emitted from thefluorescent reporter groups already incorporated into the target, whichis bound to the probe array. Probes that perfectly match the targetgenerally produce stronger signals than those that have mismatches.Since the sequence and position of each probe on the array are known, bycomplementarity, the identity of the target nucleic acid applied to theprobe array can be determined.

In other embodiments, a DNA microchip containing electronically capturedprobes (Nanogen, San Diego, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics,Nanogen's technology enables the active movement and concentration ofcharged molecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given target sequence areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

First, a test site or a row of test sites on the microchip iselectronically activated with a positive charge. Next, a solutioncontaining the DNA probes is introduced onto the microchip. Thenegatively charged probes rapidly move to the positively charged sites,where they concentrate and are chemically bound to a site on themicrochip. The microchip is then washed and another solution of distinctDNA probes is added until the array of specifically bound DNA probes iscomplete.

A test sample is then analyzed for the presence of target sequences bydetermining which of the DNA capture probes hybridize, with targetsequences. An electronic charge is also used to move and concentratetarget molecules to one or more test sites on the microchip. Theelectronic concentration of sample DNA at each test site promotes rapidhybridization of sample DNA with complementary capture probes(hybridization may occur in minutes). To remove any unbound ornonspecifically bound DNA from each site, the polarity or charge of thesite is reversed to negative, thereby forcing any unbound ornonspecifically bound DNA back into solution away from the captureprobes. A laser-based fluorescence scanner is used to detect binding.

In still further embodiments, an array technology based upon thesegregation of fluids on a flat surface (chip) by differences in surfacetension (ProtoGene, Palo Alto, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,001,311; 5,985,551; and 5,474,796; each of which is hereinincorporated by reference). Protogene's technology is based on the factthat fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction sites.For example, the A amidite is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

DNA probes unique for the target sequence (e.g., miRNA target sequence)of interest are affixed to the chip using Protogene's technology. Thechip is then contacted with the PCR-amplified genes of interest.Following hybridization, unbound DNA is removed and hybridization isdetected using any suitable method (e.g., by fluorescence de-quenchingof an incorporated fluorescent group).

In yet other embodiments, a “bead array” is used for the detection ofpolymorphisms (Illumina, San Diego, Calif.; See e.g., PCT PublicationsWO 99/67641 and WO 00/39587, each of which is herein incorporated byreference). Illumina uses a BEAD ARRAY technology that combines fiberoptic bundles and beads that self-assemble into an array. Each fiberoptic bundle contains thousands to millions of individual fibersdepending on the diameter of the bundle. The beads are coated with anoligonucleotide specific for the detection of a given SNP or mutation.Batches of beads are combined to form a pool specific to the array. Toperform an assay, the BEAD ARRAY is contacted with a prepared subjectsample (e.g., nucleic acid sample). Hybridization is detected using anysuitable method.

c. Enzymatic Detection of Hybridization

In some embodiments of the present invention, hybridization is detectedby enzymatic cleavage of specific structures.

In some embodiments, hybridization of a bound probe is detected using aTaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat.Nos. 5,962,233 and 5,538,848, each of which is herein incorporated byreference). The assay is performed during a PCR reaction. The TaqManassay exploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe, specific for a given allele or mutation, isincluded in the PCR reaction. The probe consists of an oligonucleotidewith a 5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye.During PCR, if the probe is bound to its target, the 5′-3′ nucleolyticactivity of the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dye fromthe quencher dye results in an increase of fluorescence. The signalaccumulates with each cycle of PCR and can be monitored with afluorimeter.

In still further embodiments, polymorphisms are detected using theSNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.; Seee.g., U.S. Pat. Nos. 5,952,174 and 5,919,626, each of which is hereinincorporated by reference). In this assay, SNPs are identified by usinga specially synthesized DNA primer and a DNA polymerase to selectivelyextend the DNA chain by one base at the suspected SNP location. DNA inthe region of interest is amplified and denatured. Polymerase reactionsare then performed using miniaturized systems called microfluidics.Detection is accomplished by adding a label to the nucleotide suspectedof being at the target sequence location. Incorporation of the labelinto the DNA can be detected by any suitable method (e.g., if thenucleotide contains a biotin label, detection is via a fluorescentlylabeled antibody specific for biotin).

2. Other Detection Assays

Additional detection assays useful in the detection of miRNA detectionstructures include, but are not limited to, enzyme mismatch cleavagemethods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692,5,851,770, herein incorporated by reference in their entireties);polymerase chain reaction; branched hybridization methods (e.g., Chiron,U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, hereinincorporated by reference in their entireties); NASBA (e.g., U.S. Pat.No. 5,409,818, herein incorporated by reference in its entirety);molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, hereinincorporated by reference in its entirety); E-sensor technology(Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and6,063,573, herein incorporated by reference in their entireties);cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and5,660,988, herein incorporated by reference in their entireties); DadeBehring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001,6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated byreference in their entireties); ligase chain reaction (Barnay Proc.Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridizationmethods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by referencein its entirety).

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Materials and Methods

-   -   The following final concentrations (unless noted) were used for        all reactions:    -   Probe=1 μM        INVADER=1 μM

ARRESTOR=2.67 μM

CLEAVASE XII enzyme=30 ng

All synthetic miRNA oligonucleotides were purchased from Dharmacon andgel purified on 20% denaturing acrylamide. Synthetic miRNAs were used todetermine temperature optima (see below) and LOD.

-   -   INVADER, probe, and ARRESTOR oligonucleotides were synthesized        either by Integrated DNA Technologies (IDT) or Third Wave        Technologies and purified on 20% denaturing acrylamide, unless        otherwise indicated.    -   The following 2.5× primary reaction buffer was used (unless        otherwise noted) for all reactions:

25 mM MOPS pH 7.5

62.5 mM KCl

0.125% Tween 20

0.125% Nonidet NP40

62.5 mM MgSO₄

5% PEG

-   -   Unless otherwise noted, all reactions were overlaid with 10 μl        mineral oil prior to the first thermal incubation.    -   Unless otherwise noted, synthetic miRNAs contained a 5′OH.        Experiments comparing detection of 5′ phosphorylated vs.        unphosphorylated synthetic miRNA targets indicated that there        was no significant difference in the ability of the INVADER        assay to detect these two different types of synthetic        molecules.

Example 2 Temperature Optimization Experiments for let-7 and mir-1

The oligonucleotide design for let-7 is shown in FIG. 5. Theoligonucleotide design for mir-1 is shown in FIG. 5. The followingprimary mixes were made and incubated at 50° C.±10° C. in a 96 wellplate for 30 minutes. In addition, a no target master mix was prepared(addition of H₂O in place of RNA). All reactions were covered withmineral oil to prevent evaporation.

Stock Amount Primary Reaction Components Concentration Added PrimaryReaction Buffer 2.5 X 4 μl Probe oligonucleotide (SEQ ID NOs: 10 μM 1 μl2, 6, or 9 for let 7; SEQ ID NOs: 12, 16, or 19 for miR-1) INVADERoligonucleotide (SEQ 10 μM 1 μl ID NOs: 1, 5, or 8 for let 7; SEQ IDNOs: 11, 15, or 18 for miR-1) CLEAVASE IX or XII enzyme 40 ng/μlCLEAVASE 0.5 μl   IX enzyme or 60 ng/μl CLEAVASE XII enzyme tRNA 20ng/μl 1.5 μl   Synthetic miRNA (SEQ ID NO: 4 for 100 pM 2 μl let-7a; SEQID NO: 14 for miR-1) Total 10 μl 

After completion of the primary reaction, 5 μl of the followingsecondary reaction mix were added and the reaction was then reactionincubated at 60° C. for 10-15 minutes.

Stock Amount Secondary Reaction Components Concentration Added H₂O (orbuffer for CLEAVASE 2 μl IX enzyme assays) FAM FRET probe (SEQ ID NO:21) 10 μM 1 μl Secondary Reaction Target 1.5 μM 1 μl (SEQ ID NO: 22 forlet-7; SEQ ID NO: 40 for miR-1) ARRESTOR Oligonucletide (SEQ ID 40 μM 1μl NOs: 3, 7, or 10 for let-7; SEQ ID NOs: 13, 17, or 20 for miR-1)Total 5 μl

After completion of the reaction, the plate was read in a CYTOFLUOR 4000fluorescence microplate reader using an excitation wavelength of 485 nmand emission wavelength of 530 nm. Results are shown in FIGS. 6 and 7.Stacking of the 5′-end of the INVADER oligonucleotide to the 3′-end ofthe miRNA is enhanced when the 3′-end of the INVADER oligonucleotide is2′-O-methylated. In addition, 2′-O-methylation of the 5′-end of theINVADER oligonucleotide increases the reaction temperature. Extendingthe 2′-O-methylated bases of the INVADER oligonucleotide so that theybase pair with the first two bases of the miRNA (SEQ ID NO: 8(1496-96-02) vs. SEQ ID NO: 23 (1496-96-03) in design SEQ ID NO: 9(1496-96-01R) of let-7a) increases the temperature optimum of thedescribed reaction but does not enhance the detection.

Example 3 LOD Experiments for let-7 and miR-1

After determining the optimal reaction temperature for each set of probeand INVADER oligonucleotides and determining the best working design(from the temperature optimization net signal), the following experimentwas set up to determine the LOD of the design using synthetic RNA. Thefollowing reaction mix was aliquoted into a 96-well plate (see platesetup below) with each well containing:

Component Stock conc. Amount Added Primary reaction buffer 2.5 X 4 μlProbe 10 μM 1 μl SEQ ID NO: 6 for let 7 SEQ ID NO: 16 or 19 for miR-1INVADER oligo 10 μM 1 μl SEQ ID NO: 5 for let 7 SEQ ID NO: 15 or 18 formiR-1 CLEAVASE XII enzyme 60 ng/μl 0.5 μl   TRNA 20 ng/μl 1 μl TOTAL 7.5μl  2.5 μl of the following miRNA concentrations were added in triplicatesor quadruplicates using the following setup:

<-------[miRNA]----> 1 nM 100 pM 10 pM 1 pM 100 fM 10 fM H2O A B C DThe plate was overlayed with mineral oil (10 μl) and incubated at 50° C.for 2 hrs. After completion of the primary reaction, 5 μl of thefollowing was added to each well and the plates were incubated at 60° C.for 1.5 hrs. The plate was read using the settings described above (seeExample 2).

Secondary Reaction Components Stock Conc. Amount Added H₂O (or bufferfor 2 μl CLEAVASE IX enzyme assays) FAM FRET Probe (SEQ ID 10 μM 1 μlNO: 21) Secondary Target (SEQ ID 1.5 μM 1 μl NO: 22 for let-7; SEQ IDNO: 40 for miR-1) ARRESTOR 40 μM 1 μl oligonucleotide (SEQ ID NO: 7 forlet 7; SEQ ID NOs: 17 or 20 for miR-1) Total 5 μl

The LOD for let-7 and mir-1 was next tested on human RNA samples. Theprotocol described above was utilized. 50-100 ng of tissue specifictotal human RNA samples (Clonetech, Palo Alto, Calif.) was used. Resultsare shown in FIGS. 8 and 10. Using total RNA the let-7a INVADER assaydetects the same tissue expression profile as seen before for let-7aexpression levels depending on the source of tissue (Pasquinelli et al.,408:86 [2000]).

Example 4 Cross Reactivity Experiments for let-7 a,c,e, and f

This Example describes an analysis of the cross reactivity of probeand/or INVADER oligonucleotides directed against one sub-type of let-7for another sub-type. The protocol for synthetic let-7a miRNA setupdescribed in Example 3 was utilized. FIG. 5 shows the oligonucleotidedesigns. The following plate setup was used:

10 1 100 10 1 100 10 H2O nM 1 nM 2 pM 3 pM 4 pM 5 fM 6 fM 7 8 Let7A ALet7A B Let7C C Let7C D Let7E E Let7E F Let7F G Let7F H

The results are shown in FIG. 9. For let-7a design, cross reactivity ismaximum when the miRNA is of the same length with a one base change awayfrom the cleavage site. In other words, mismatches at the INVADERoligonucleotide/miRNA hybridizing regions result in high crossreactivity when the mismatch is furthest from the cleavage site(let-7c). Cross reactivity is the lowest when base changes are opposite(or close to) the cleavage site. For let-7a, the worst cross reactivityis with let-7c, which results in 25% of the signal. This Exampledemonstrates that the INVADER assay is able to differentiate betweenvery similar miRNAs.

Example 5 CLEAVASE IX Enzyme vs CLEAVASE XII Enzyme

This Example describes the optimization of CLEAVASE enzymes for use inmiRNA assays. The protocol for temperature optimization described abovewas utilized. Either 20 ng of the CLEAVASE IX enzyme (Third WaveTechnologies, Madison, Wis.) or 30 ng of the CLEAVASE XII enzyme (ThirdWave Technologies, Madison, Wis.) was used. The following buffer wasused for the CLEAVASE IX enzyme:

2.5× primary reaction buffer: 25 mM MOPS pH 7.5, 250 mM KCl, 0.125%Tween 20,

0.125% Nonidet NP40, 31.25 mM MgSO₄, 10% PEG.

7.5× secondary reaction buffer: 87.5 mM MgSO₄

The following buffer was used for the CLEAVASE XII enzyme:

2.5× primary reaction buffer: 25 mM MOPS pH 7.5, 62.5 mM KCl, 0.125%Tween 20,

0.125% Nonidet NP40, 62.5 mM MgSO₄, 5% PEG.

7.5× secondary reaction buffer: H₂O

The LOD experimental protocol was used with either the CLEAVASE IX orXII enzymes. The LOD was determined for both enzymes. The results areshown in FIG. 11.

Signal increased linearly with increasing amounts of the let-7 miRNAwhen assayed with either the CLEAVASE IX enzyme or the CLEAVASE XIIenzyme. However, R² values were greater in the CLEAVASE XII enzyme,indicating greater linearity. Moreover, the LOD was lower with theCLEAVASE XII enzyme. The net signal for the detection of 2.5 amoles was20 counts with the CLEAVASE IX enzyme and 66.75 with the CLEAVASE XIIenzyme.

Example 6 miR-135, GAPDH and U6 RNA

A. Design of Oligonucleotides to Detect miR135

This example describes assay design and LOD determination for miRNAmiR135. Experiments were performed as described in Examples 2 and 3 formiR-1. The oligonucleotide designs are described in FIG. 5. Each of thedesigns (A-D) utilizes different INVADER and probe oligonucleotides forthe detection of mir-135 miRNA. Results of the temperature optimizationexperiments comparing performance of all of the designs are shown inFIG. 13. Design D gave the highest signal. Results of LOD experimentsusing assay design D are shown in FIG. 14.

FIG. 14A presents the raw counts generated from four replicate assays ateach of the indicated target concentrations. The average counts obtainedwith each target concentration are indicated as are the net signal andfold-over-zero (FOZ). The limit of detection of the miR-135 target inthis experiment was 164 zmoles, equivalent to 98,743 molecules. FIG. 14Bcontains a graphical representation of the average counts obtained ateach concentration and indicates that the INVADER assay is linearthroughout much of the concentration range tested.

B. Design of Oligonucleotides to Detect GAPDH and U6 RNA

In some circumstances, it may be desirable to co-detect, e.g. in abiplex assay, an RNA generally present in all cells at constant levelsalong with one or more miRNA species, which may be expressed in atissue-specific manner. INVADER assays were therefore designed to twodistinct RNAs generally found in all cell types: humanglyceraldehydes-3-phosphate dehydrogenase (hGAPDH) and U6 RNA.

In the case of hGAPDH, the following oligonucleotides have been used inbiplex miRNA detection assays: INVADER oligonucleotide (SEQ ID NO: 41);probe (SEQ ID NO: 42); ARRESTOR oligonucleotide (SEQ ID NO: 43); SRToligonucleotide (SEQ ID NO:49), FRET oligonucleotide (red dye) (SEQ IDNO: 48).

In the case of U6, sequence alignments of the U6 RNAs of 8 diversespecies from C. elegans to mouse to arabidopsis to humans to identify aregion suitable for the design of a “universal” INVADER assay. Thealignment is shown in FIG. 12; the oligonucleotide sequences created todetect this sequence are SEQ ID NOs: 93-95.

Initial experiments carried out with these oligonucleotides on celllysates using SEQ ID NOs: 45-47 demonstrated that signal from U6reactions reached saturation well before miRNA signal, possibly owing tolarge quantities of U6 RNA in cells. Therefore, titration reactions werecarried out to determine whether diluting the probe and INVADERoligonucleotide concentrations would render this probe set suitable foruse in biplex miRNA detection assays with INVADER and probe finalconcentrations ranging from 1 μM to 12.5 nM. Final concentrations of theINVADER and probe oligonucleotides between 12.5-50 nM were suitable forbiplex miRNA detection for miR-1d and let-7a. ARRESTOR, SRT, and FRETprobe concentrations were as described in the previous examples. Furtherexperiments demonstrate that detection of U6 RNA with the “universal” U6RNA oligonucleotides (SEQ ID NOs: 93-95) is comparable to detection withSEQ ID NOs: 45-47.

Example 7 Detection of let-7, GAPDH, and U6 RNA in Cell Lysates

A. Detection of let 7a in Cell Lysates

This example describes detection of the let-7 miRNA directly in totalcell RNA as well as in uninduced fibroblast cells from a humanosteosarcoma cell line, MG63 (Third Wave Technologies, Madison, Wis.;catalog number CRL-1427). Total cell RNA was extracted using TRIZOL(Gibco-BRL), as previously described (Chomczynski et al., Anal. Biochem.162: 156-156 (1987)), and cell lysates were prepared as described by Eiset al., Nature Biotechnology, 19: 673-6 (2001); both publications areherein incorporated by reference.

Reactions were set up as follows. Aliquots of 5 μl of either celllysate, synthetic miRNA target in lysis buffer (Eis et al., NatureBiotechnology, 19: 673-6 (2001)) at the indicated concentrations, or 5μl of 20 ng/μl of tRNA (for the no target controls) were pipetted intothe appropriate wells of a microtiter plate. A primary reaction mastermix was made for 96 reactions containing the following reagents.

Amount per Total added Stock reaction to Master Reagent concentration(μl) Mix (μl) Mixture of Probe Probe 20 μM/ 0.5 45 oligonucleotideINVADER 1496-78-01 R (SEQ oligonucleotide ID NO: 6) and 200 μM INVADERoligonucleotide 1496-78-02 (SEQ ID NO: 5) CLEAVASE XII 60 ng/μl 0.5 45enzyme Primary Buffer 2.5 X 4 360 TOTAL 5 450

Aliquots of 5 μl of the primary reaction master mix were added to thewells containing the appropriate target or control. The plate wasoverlayed with mineral oil (10 μl) and incubated at 53° C. for 2 hrs.After completion of the primary reaction, 5 μl of the following wasadded to each well, and the plates were incubated at 60° C. for 1.5 hrs.The plate was read using the settings described above (see Example 2).

Secondary Reaction Components Stock Conc. Amount Added H2O (or bufferfor CLEAVASE 2 μl IX enzyme assays) FAM FRET probe (SEQ ID NO: 10 μM 1μl 21) Secondary Reaction Target (SRT) 1.5 μM 1 μl SEQ ID NO: 22ARRESTOR oligonucleotide SEQ 40 μM 1 μl ID NO: 7 Total 5 μl

All targets were assayed in quadruplicate. The average counts obtainedfor the different numbers of cells assayed for both total RNA and celllysates were plotted in FIG. 15. A standard curve obtained from INVADERassays on known quantities of synthetic let-7a miRNA was used toextrapolate the let-7a copy number per cell. The number of cells fromwhich cell lysates were generated was determined during the seedingprocedure prior to cell lysis as described in Eis et al., NatureBiotechnology, 19: 673-6 (2001), herein incorporated by reference. Inthis experiment, the limit of detection in cell lysates was reached inthe total RNA extracts obtained from 156 cells.

B. Lysis in Absence of Mg⁺⁺

An alternative lysis procedure was developed as follows. It had beennoted that when the above lysis procedure was used, long mRNAs, i.e.from GAPDH, were not being detected in the quantities expected.Experiments were carried out to examine the effect of Mg⁺⁺ on extractionof RNA in lysates. Extracts lysed in the presence or absence of MgCl₂were compared to total cell RNA extracts prepared using TRIZOL asdescribed above in this example.

Hela cells (7.5×10⁶ cells) were suspended in 100 μl of a solution of 10mM MOPS buffer, pH 7.5, with 100 mM KCl. Aliquots of 10 μl were added toseparate tubes and lysed with 100 μl of two different lysis buffersprepared as follows:

MOPS lysis w/Mg⁺⁺ MOPS lysis w/out Mg⁺⁺ 180 μl 11 μg/ml tRNA 180 μl 11μg/ml tRNA 0.5 ml NP40 0.5 ml NP40 4 ml 0.5M MOPS 4 ml 0.5M MOPS 0.5 ml1M MgCl₂ N/A 4.82 ml H₂O 5.32 mls H₂O 10 mls 10 mls

All tubes were then incubated at 80° C. for 15 minutes to lyse thecells, and then centrifuged to pellet debris. Aliquots of 5 μl of thevarious lysates were added to INVADER reactions as follows.

Primary INVADER reactions were as described above for let-7a; PIoligonucleotide mixes were also made for GAPDH (SEQ ID NOs: 41-43) andfor U6 (SEQ ID NOs: 45-47 at 50 nM final concentration).

Amount added Final Component per reaction concentration PIoligonucleotide mix* 0.25 μl   1 μM each* H₂O 0.25 μl   0.25 μl  CLEAVASE XII enzyme 0.5 μl   0.5 μl   60 ng/μl 4 μl 4 μl Total 5 μl 5 μl*Primary INVADER reactions were as described above for let-7a; PIoligonucleotide mixes were also made for GAPDH (SEQ ID NOs: 41-43) andfor U6 (SEQ ID NOs: 45-47 at 50 nM final concentration).Primary reaction mixtures were incubated at 49° C. for 1 hour. Aliquotsof the following secondary reaction mixture were then added:

Component Amount added per reaction Secondary reaction mixture* 1.5 μlH₂O 3.5 μl Total   5 μl

Secondary reaction mixture included SRTs (SEQ ID NO: 22 for let-7; SEQID NO: 49 for GAPDH and U6) target, FRET oligonucleotides (SEQ ID NO: 21for let-7; SEQ ID NO: 48 for GAPDH and U6), arrestors (SEQ ID NO:7 forlet-7, SEQ ID NO:43 for GAPDH, and SEQ ID NO: 47 for U6) at theconcentrations indicated in Example 7A.

Secondary reactions were run at 60° C. for 1 hour. Reactions were readon a CYTOFLUOR microplate reader as described in Example 2. The resultsare presented in FIG. 16 and indicate that presence of the GAPDH signalis dependent on the absence of Mg++ from the lysis buffer, whereas U6RNA signal remains relatively constant regardless of the presence ofMg++. Additional experiments confirmed that all RNAs were detectable intotal cell RNA at levels comparable to those obtained from lysis in theabsence of Mg++.

Example 8 Alternative INVADER Assay Designs for Detection of VariousmiRNAs

A. Alternative Designs for Detection of let-7A

This example describes the creation and testing of alternativeoligonucleotide designs for detection of the let-7a miRNA. In one seriesof experiments, a set of alternative designs was created in which thetarget specific regions of both the INVADER oligonucleotide and theprobe oligonucleotide were eleven nucleotides long. A second set ofdesigns was created in which the target specific regions of the probeoligonucleotides were 10 nucleotides long and the target specificregions of the INVADER oligonucleotides were 12 nucleotides long.

1. Oligonucleotide Designs

a. 11-mer Probe and INVADER Oligonucleotide Designs

FIG. 5 shows sets of alternative oligonucleotide designs for detectionof the let-7a miRNA in which the target specific regions of both theprobe and INVADER oligonucleotides are 11 nucleotides long. SEQ ID NOs:50-51 provide a design in which both the INVADER and probeoligonucleotides are linear. SEQ ID NO: 6 contains a probeoligonucleotide that forms a stem-loop structure); SEQ ID NO: 5, anINVADER oligonucleotide that forms a stem-loop structure); SEQ ID NOs:5-6, both probe and INVADER oligonucleotides with stem-loops.

b. 10-mer Probe and 12-mer INVADER Oligonucleotide Designs

FIG. 5 shows a set of alternative oligonucleotide designs for detectionof the let-7a miRNA in which the target specific regions of the probecomprise 10 nucleotides, and those of the INVADER oligonucleotides, 12nucleotides. SEQ ID NOs: 52-53 provide a design in which both theINVADER and probe oligonucleotides are linear. SEQ ID NO: 2 contains aprobe oligonucleotide that forms a stem-loop structure); SEQ ID NO: 1,an INVADER oligonucleotide that forms a stem-loop structure).

2. Temperature Optimization Profiles of Alternative OligonucleotideDesigns for Detection of the let-7a miRNA

Temperature optimization experiments were carried out as follows. Amaster mix was made for 24 reactions. Each reaction contained thefollowing:

Volume per Final Stock concentration reaction concentration 2.5 XPrimary reaction buffer for 4 μl 1 X the CLEAVASE XII enzyme (asdescribed in Example 5) 10 μM probe* 1 μl 1 μM 100 μM INVADERoligonucleotide* 1 μl 10 μM 60 ng/μl CLEAVASE 12 0.5 μl   30 ng H₂O 2.5μl   N/A 30 pM miRNA (for the 11-mer 1 μl 3 pM OR 1 nM temperatureoptimizations) OR 10 nM miRNA (for the 10-mer probe/12-mer INVADERoligonucleotide temperature optimizations) 20 ng/μl tRNA (for no target1 μl 2 ng controls only) TOTAL 10 μl  *Various combinations of probe andINVADER oligonucleotides were used in this experiment as indicated inFIGS. 16-17.

Secondary reaction mixes were as described in Example 3 for let-7. Whereappropriate, ARRESTOR sequences were made to compliment the entire loopand target specific regions of the probe and extending 6 bases towardthe 5′ end of the probe.

In the case of the 11-mer temperature optimization experiment, theprimary reactions were run at 50±9° C. for 1 hour followed by a 15minute secondary reaction at 60° C. as described in Example 2. As forthe 10-mer probe, with the 12-mer INVADER oligo, the primary reactionswere run at 50±9° C. for 15 minutes followed by a 15 minute secondaryreaction at 60° C.

Results for the designs in which the target specific portions of theINVADER and probe oligonucleotides were 11 nucleotides long arepresented in FIG. 18. FIG. 18A shows the temperature optimizationprofiles of each design. FIG. 18B shows the normalized maximumperformance of each design, including the optimum temperature for each.Results for the designs in which the target specific portion of theprobe oligonucleotide was 10 bases and that of the INVADERoligonucleotide was 12 are presented in FIG. 19. FIG. 19 A shows thetemperature optimization profiles, and FIG. 19 B, the normalized maximumperformance of each design.

Examination of these results suggests that which design results inmaximum performance varies depending on both reaction conditions and therelative stability of the miRNA-oligonucleotide hybrid formed. Forexample, when the target specific regions of both oligonucleotides are11 bases long, the probe target specific region has a predicted Tm of49° C. and that of the INVADER, of 37° C. In this case, stabilization ofthe INVADER oligonucleotide-miRNA interaction confers improved assayperformance on this design. However, for the let-7a designs in whichprobes were 10-mers and INVADER oligonucleotides, 12-mers, the targetspecific regions of the two oligonucleotides have approximatelyequivalent Tms. In this case, the design in which both oligonucleotidesare looped works best.

3. LOD of let-7a Using Two Alternative Designs

Experiments were set up as described in Example 3 to compare the LOD ofthe double loop design and the single loop design, in which the INVADERoligonucleotide forms a stem-loop structure.

Reactions to determine LOD were run in quadruplicate. Reaction mixturescontained the following reagents (final concentrations):

Volume per Final Stock concentration reaction concentration 2.5 XPrimary reaction 4 μl 1 X buffer for CLEAVASE XII enzyme 10 μM probe/200μM 0.5 μl   1 μM probe/20 μM INVADER INVADER oligonucleotide mixoligonucleotide (sequences as indicated in FIG. 20) 60 ng/μl CLEAVASEXII 0.5 μl   30 ng enzyme Total 5 μl

Aliquots of 5 μl of miRNA were added to the wells containing thereaction mixtures at the final concentrations indicated in FIG. 20.Primary reactions were run for 1.5 hours at the optimal temperatures foreach designed as determined in Example 8B (50° C. for the looped INVADERoligonucleotide design and 53° C. for the double loop design). Thesecondary reactions were set up as described in Examples 2 and 3 and runfor 1 hour at 60° C.

The results in FIG. 20 show net signal produced as a function of molesof miRNA. The linear ranges of the plots indicated that more signal wasproduced from a given amount of miRNA using the INVADER loop design thanfrom the double loop design. Similarly, an examination of the table inFIG. 20 indicates that the fold-over-zero values at each miRNA level aregreater for the single loop design. Both designs resulted in sufficientFOZ at the lowest concentrations tested, 2.68×10⁻²⁰ moles, or 26.8zeptomoles, equivalent to approximately 16,000 molecules.

4. Full Length Vs. Shortened ARRESTOR Oligonucleotides

Experiments were conducted to evaluate the relative performance offull-length ARRESTOR molecules, e.g. as shown in FIGS. 4 and 12, inwhich the ARRESTOR molecules extend at their 5′ ends around the loop,throughout the length of the miRNA-specific region of the probe and intothe 5′ flap region vs. shortened ARRESTOR molecules that arecomplementary only to the miRNA-specific region of the probe and part ofthe 5′ flap but do not extend into the loop region or beyond. Reactionswere set up as follows to detect synthetic let-7a miRNA:

Stock Amount added Component concentration per reaction PI mix (probeSEQ ID NO: 10 μM probe 1 μl 6; INVADER 50 μM INVADER oligooligonucleotide SEQ ID NO: 5) CLEAVASE XII enzyme (60 ng/μl) 0.5 μl  H₂O 0.5 μl   Primary Reaction Buffer 2.5 X 4 μl Total 6 μl

Aliquots of 6 μl of the primary reaction mix were added to theappropriate wells of a microtiter plate followed by aliquots of 4 μl ofsyntheticlet-7a miRNA or 4 μl of 10 ng/μl tRNA in dH₂O at the finalconcentrations indicated in the table below. Primary INVADER reactionswere incubated at 53° C. for 1.5 hours.

Aliquots of secondary reaction mixtures were added as follows:

Full-length ARRESTOR Stock Component concentration Amount added ARRESTOR40 μM 1 μl SEQ ID NO: 7 for full length ARRESTOR, SEQ ID NO: 54 forshortented ARRESTOR MO5 SRT 1.5 μM 1 μl (SEQ ID NO: 22) FRET FAM (SEQ ID10 μM 1 μl NO: 21) H₂O 2 μlSecondary reactions were incubated at 60° C. for 1.5 hours. Microtiterplates were read as described in Example 2. The results were as shown inFIG. 17.

These results indicate that there is no significant different in signalgeneration or limit of detection when full-length or shortened ARRESTORoligonucleotides complementary to the miRNA-specific portion of theprobe are used in the secondary INVADER reaction.

B. Alternative Designs Using Linear Probe and INVADER Oligonucleotides

Alternative designs were tested in which both the probe and INVADERoligonucleotides contain a universal sequence, and neitheroligonucleotide forms a hairpin. A schematic of the design is presentedin FIG. 4. The universal sequence is present on the 5′ end of theINVADER oligonucleotide and on the 3′ end of the probe oligo. A short,complementary “capture” oligonucleotide is added and is comprised of2′-O-methyl residues, allowing it to promote co-axial stacking in thepresence of the miRNA (e.g. SEQ ID NO: 60). Designs were created forboth miR-15 (SEQ ID NOs: 58-59 and 61) and mir-135 (SEQ ID NOs: 63-65).Initial designs, while leading to high non-specific background signal inthe absence of miRNA target, nonetheless indicate that it is feasible todetect miRNAs with such universal capture oligonucleotides.

Example 9 Effect of 2′-O-Methylation of Nucleotide Residues in the Loops

This example describes experiments aimed at assessing the effect ofsubstituting 2′-deoxy residues for some or all of the 2′-O-methylresidues incorporated in the probe and INVADER oligonucleotides used fordetecting miRNAs. All of the designs presented in the preceding examplesinclude 2′-O-methyl residues in the loop regions as described in Example2. Experiments were conducted to test the effect of substituting 2′deoxy residues for some or all of the 2′-O-methyl residues in theINVADER and probe oligonucleotides designed to detect the let-7a miRNA.

FIG. 5 shows the modified let-7a designs. SEQ ID NOs: 5-6 contain2′-O-methyl residues as described in Example 2. The design in SEQ IDNOs: 73-74 contain 2′deoxy residues at all positions; and those in SEQID NOs: 75-76, 2′-O-methyl residues in the portions of the stemsadjacent to the target.

INVADER reactions were set up to compare the signal generation andtemperature optima of the three different designs. Reactions were asdescribed in the LOD experiments in Example 8 and included 100 μMsynthetic miRNA, 1 μM probe, and 10 μM INVADER oligonucleotide. Primaryreactions were run for 15 minutes at the temperatures indicated;secondary reactions were run for 5 minutes at 60° C.

The results of the INVADER assays are shown in FIG. 21 and indicate thatthe design in which the stem loop structures are comprised of2′-O-methyl residues yields the most signal, followed by the design inwhich the bases adjacent to the target are comprised of 2′-O-methylresidues. The oligonucleotides comprised entirely of 2′-deoxy residuesgenerated the lowest levels of signal.

A further set of experiments was designed to test additional designvariations as follows: probe and INVADER oligonucleotides with shorterhairpins, probe and INVADER oligonucleotides with more stable loops or,alternatively with shorter loops, probe and INVADER oligonucleotideswith only three 2′-O-methyl residues. Primary reactions were set up totest detection of miR-15 as follows.

The following probe/INVADER oligonucleotide combinations were tested.

Probe INVADER oligo 1544-71-01 SEQ ID NO: 55 1544-71-02 SEQ ID NO: 561544-71-01 SEQ ID NO: 55 1796-43-02 SEQ ID NO: 68 1544-71-01 SEQ ID NO:55 1796-43-04 SEQ ID NO: 70 1544-71-01 SEQ ID NO: 55 1796-43-06 SEQ IDNO: 72 1796-43-01 SEQ ID NO: 67 1544-71-02 SEQ ID NO: 56 1796-43-03 SEQID NO: 69 1544-71-02 SEQ ID NO: 56 1796-43-05 SEQ ID NO: 71 1544-71-02SEQ ID NO: 56 1796-43-03 SEQ ID NO: 69 1796-43-04 SEQ ID NO: 70

Primary reaction mixes were made as follows.

Stock Primary Reaction Component Concentration Amount Added Probeoligonucleotide (as 40 μM 0.25 μl   indicated in above table) INVADERoligo 40 μM 0.25 μl   CLEAVASE XII enzyme 60 ng/μl 0.5 μl   PrimaryReaction Buffer 2.5 X 4 μl Total 5 μl

Aliquots of 5 μl of Primary reaction mix were added to 5 μl of syntheticmiR-15 RNA at the following final amounts: 0, 0.1 amole, 0.33 amole,1.09 amole. Primary reactions were incubated at 52.5° C. for 2 hours.

Secondary reaction mixes were made as follows.

Secondary reaction component Concentration Amount Added FAM FREToligonucleotide 13.4 μl FAM FRET 0.75 μl (SEQ ID NO: 21) and 2 μM SRTSecondary Reaction Target (SRT) (SEQ ID NO: 40) ARRESTOR 54 μM 0.75 μloligonucleotide (SEQ ID NO: 66) H₂O  3.5 μl Total   5 μl

Aliquots of 5 μl were added and the reactions incubated at 60° C. for 45minutes. The results, in relative fluorescent units (RFUs) are presentedbelow.

Probe 1544-71-01 1544-71-01 Invader 1544-71-02 NET FOZ 1796-43-02 NETFOZ 1.09  655 658 662 566 7.13 1.09 706 738 777 636 7.12 amole amole0.33  314 262 256 185 3.00 0.33 281 287 290 182 2.75 amole amole 0.10 122 138 134 39 1.42 0.10 149 150 153  47 1.45 amole amole 0 amole  8893 96 0 amole   104 101 107 Probe 1796-43-01 1796-43-03 Invader1544-71-02 NET FOZ 1544-71-02 NET FOZ 1.09 1689 1744 1895 146 1.09 1.09882 869 847 702 5.27 amole amole 0.33 1655 1717 1817 99 1.06 0.33 335341 341 175 2.06 amole amole 0.10 1692 1693 1695 63 1.04 0.10 196 209196  36 1.22 amole amole 0 amole 1636 1601 1654 0 amole   169 165 1591544-71-01 1544-71-01 1796-43-04 NET FOZ 1796-43-06 NET FOZ 1.09  676688 693 579 6.43 1.09 625 562 579 501 6.69 amole amole 0.33  274 275 264164 2.54 0.33 229 215 204 128 2.45 amole amole 0.10  153 137 143 38 1.350.10 126 121 112  32 1.36 amole amole 0 amole  111 107 102 0 amole    94 87  83 1796-43-05 1796-43-05 1544-71-02 NET FOZ 1796-43-06 NET FOZ 1.09 806 824 773 708 8.64 1.09 772 752 704 631 6.65 amole amole 0.33  280280 262 181 2.96 0.33 260 252 251 143 2.28 amole amole 0.10  144 145 13950 1.54 0.10 140 142 139  29 1.26 amole amole 0 amole  91 95 92 0amole   115 109 111

These results suggest that the designs in which the probeoligonucleotide contained a shortened hairpin and a highly stabletetra-loop comprised of 2′-O-methyl residues in combination with theoriginal INVADER oligonucleotide design (2′-O-methyl residues, TTTTloop, long hairpin) may generate a somewhat higher FOZ value. Otherwise,none of the alternative design oligonucleotide sets offered anyimprovement over the original designs. It is noteworthy that thecombination of an all-DNA INVADER oligonucleotide with the originalchimeric probe oligonucleotide gave FOZ values approximately equivalentto those obtained with both chimeric probe and INVADER oligonucleotides.In some applications, substitution of an all DNA INVADER oligonucleotidemay be desirable to reduce oligonucleotide synthesis costs and may bemade without sacrificing limit of detection.

Further experiments demonstrated that it is possible to compensate forsub-optimal signal generation with particular oligonucleotide sets byadding more RNA (e.g. lysate, purified total RNA, synthetic miRNA) tothe reaction. Similarly, additional experiments in which variousoligonucleotides (i.e. probe, INVADER, ARRESTOR, or various combinationsthereof) were gel purified as described in Example 1 indicated thatstandard gel purification of all three types of oligonucleotides givesmaximal signal. It is possible to achieve signal levels approximatelyequal to the maximal levels with gel purified probes if the otheroligonucleotides, i.e. the INVADER and ARRESTOR oligonucleotides, aredesalted following synthesis.

Example 10 Detection of miRNA Expression in Total RNA from MultipleTissue Types

This example describes experiments carried out to test the suitabilityof the INVADER assay to detect different miRNA species in total RNAextracted from diverse tissue types. In order to evaluate tissuespecific gene expression, temperature optima and LODs were firstdetermined for each design.

1. INVADER and Probe Oligonucleotide Designs

INVADER assay oligonucleotides were designed to detect the miR-15,miR-16, and miR-125b miRNA species. The designs for these assays arepresented in FIG. 5. The designs for let-7a and miR-135 are described inExamples 2 and 6, respectively.

2. Determination of Temperature Optima and LODs

Temperature optimization experiments were conducted for each of theseoligonucleotide sets as described in Example 8. Each primary reactionincluded 1 nM of the targeted miRNA and was carried out for 15 minutesat temperatures ranging from 50±9° C. Secondary reactions were asdescribed in Example 2 and were run for 1 to 1.5 hours at 60° C. Optimumtemperatures were as follows:

let-7a 53° C. miR-15 53° C. miR-16 56° C. MiR-125b 52° C. MiR-135 45° C.

Once the temperature optima were obtained, LODs were determined for eachmiRNA species as described in Example 8. All LODs were ≦30 zeptomoles.

3. Gene Expression Profiling

Gene expression profiling was carried out on total RNA extracted from 20different tissue types. Total RNA was purchased from Clontech (PaloAlto, Calif., catalog number K4008-1, Human Total RNA Master Panel II).For let-7a, 50 ng of total RNA was tested in each reaction; for theother miRNA species, 100 ng of total RNA was tested. All reactions wereset up as described in Example 8; primary reactions were run at thetemperature optima for 1.5 hours; secondary reactions were as describedabove. The gene expression profiles for each miRNA species are presentedin FIG. 23. These results indicate that the INVADER assay can be used toexamine miRNA expression in different tissue types. These data furthersuggest that let-7a and miR-125b are expressed in a wide variety oftissues; the other miRNA species appear to be more specific to a limitednumber of tissue types.

Example 11 Effects of Variable Oligonucleotide Length on INVADER AssayDetection of miRNA

This example describes the impact of alterations in probe and INVADERoligonucleotide length on detection of the let-7a 22-nt miRNA. Inparticular, these experiments compare detection of an miRNA that formsperfect stacking interactions between the ends of the probe and INVADERoligonucleotides to detection of an miRNA that forms both 5′ and 3′overlaps as well as to one that results in a single nucleotide gap atboth the 5′ and 3′ ends.

FIG. 24 shows the results of analyzing three different types of designs.SEQ ID NOs: 5-6 shows a perfect stack between the 22-nt target and theflanking ends of the looped probe and INVADER oligonucleotides. In SEQID NOs: 83-84, both the probe and INVADER oligonucleotides are extendedby a single base, resulting in both 5′ and 3′ overlaps. In SEQ ID NOs:85-86, both the probe and INVADER oligonucleotides are shortened by asingle base, relative to the designs in SEQ ID NOs: 5-6, resulting in asingle nucleotide gap at both ends.

INVADER assays were set up to test the performance of theseoligonucleotide sets for detection of synthetic let-7a miRNAs. Reactionswere carried out as described in Example 8 and included 100 μM syntheticlet-7a miRNA, 1 μM probe and 10 μM INVADER oligonucleotide. Primaryreactions were run for 15 minutes at 53° C.; secondary reactions, for 5minutes at 60° C., as described in Example 2. The results are presentedin FIG. 24.

These data indicate that in this experiment, a single nucleotide overlapat both ends of the miRNA target resulted in an approximately 30%decrease in signal generation as well as a reduction of 2° C. in optimaltemperature. A one nucleotide gap at both ends of the target, however,did not reduce signal generation, though it did reduce the optimalreaction temperature by 5° C.

Example 12 Discrimination of miRNA from Precursor RNA and from EncodingDNA

Experiments were carried out to determine whether the INVADER miRNAassay discriminated the miRNA target itself from both its precursor RNAand from the DNA encoding the miRNA.

A. Precursor Cross-Reactivity Test

Precursor let-7 RNA (SEQ ID NO: 87) was transcribed in vitro andanalyzed by capillary electrophoresis to determine whether it containedany fragments that might mimic the let-7a miRNA. The shortestcontaminating fragment was estimated to be approximately 45 nt. LODreactions were run essentially as described in Example 3 at precursor orsynthetic 5′ P let-7a mi-RNA concentrations as indicated in the tablebelow. PI mixes contained 10 μM probe SEQ ID NO: 6 and 100 μM INVADERoligonucleotide SEQ ID NO: 5. Primary reactions were run at 53° C. for 1hour; secondary reactions were run at 58° C. for 1 hour with secondaryreaction mixes essentially as described in Example 3 (FRET probe SEQ IDNO: 21, SRT SEQ ID NO: 22, and ARRESTOR SEQ ID NO: 7). The results ofthis experiment indicated that this miRNA assay is approximately 4%cross reactive vs. the precursor RNA.

B. Discrimination of RNA Vs. DNA Signal

Reactions were run to detect let-7a miRNA in cell lysates as describedin Example 7. Prior to detection with the INVADER assay, aliquots 1 μmof 8 μg/μl RNAse A (Qiagen, Inc.) were added to 80 μl of cell lysate andincubated at 37° C. for 2.25 hours. The RNAse A treated samples failedto generate any signal above background, indicating that signalgenerated in assays lacking RNAse A arises from detection of the miRNAtarget and not the encoding DNA (FIG. 16). Further experiments werecarried out in which RNAse A was added either prior to the primaryreaction or prior to the secondary reaction. When RNAse A was addedprior to the primary reaction, no signal was generated, consistent withthe previous results. When RNAse A was added subsequent to the primaryreaction, no loss of signal was observed, further indicating that thesignal being detected is due to RNA and that there is no adverse effectof RNAse on other reaction components, e.g. the CLEAVASE enzyme.

Example 13 Detection of a Dual form miRNA

Oligonucleotide designs were created for miR-124a. Theseoligonucleotides can be used to detect two naturally occurringmiRNAs—one 21 nt in length and the other, 22 nt.

Temperature optimization reactions were set up, essentially as describedin Example 3, using 1 nM of synthetic miRNA target, 25 primary reactionand a 15 minute secondary reaction. The oligonucleotides used in thesereactions are listed in FIG. 5 (SEQ ID NOs: 90-92). Temperature profilesfor the two different length miRNA targets are shown in FIG. 22 andindicate that the same oligonucleotide designs can be used to detectboth targets.

Example 14 Oligonucleotide Designs for Detection of an siRNA

Approaches similar to those described in the preceding examples maysimilarly be used to detect siRNAs. FIG. 25 illustrates two alternativeINVADER assay designs for detection of a β-actin siRNA. This siRNA isdescribed in Harborth, J. et al., Journal of Cell Science, 114:4557-4565 (2001). One design is presented for each the sense andantisense strands; exemplary oligonucleotides for detecting this siRNAare listed in FIG. 26, SEQ ID Nos: 101-106.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology, genetics, or related fields are intended to be withinthe scope of the following claims.

The invention claimed is:
 1. A method for analyzing microRNA in asample, comprising: a) hybridizing a microRNA target to at least one DNAnucleic acid, wherein said DNA nucleic acid contains a first region thatis complementary to at least a portion of said microRNA target, and asecond region that is not complementary to said microRNA target, whereinsaid second region of said nucleic acid forms a hairpin loop to form ahalf dumbbell structure when said DNA nucleic acid is hybridized to saidmicroRNA, to generate a detection structure; and b) detecting saiddetection structure.
 2. The method of claim 1, further comprisingreacting said detection structure with at least one of astructure-specific nuclease or a DNA polymerase to form a modifieddetection structure, wherein said reacting comprises cleaving and/orextending at least one of said DNA nucleic acid or said microRNA in saiddetection structure.
 3. The method of claim 1, wherein said detectingcomprises quantitating said microRNA.
 4. The method of claim 1, whereinsaid detecting comprises forming an invasive cleavage structure, anddetecting the cleavage of said invasive cleavage structure.
 5. Themethod of claim 4, wherein said detection structure comprises first andsecond oligonucleotides configured to form an invasive cleavagestructure in combination with said microRNA, wherein formation of aninvasive cleavage structure with said microRNA requires both of saidfirst and said second oligonucleotides.
 6. The method of claim 4,wherein said detection structure comprises a DNA first oligonucleotideconfigured to form an invasive cleavage structure in combination withsaid microRNA, wherein said DNA first oligonucleotide and said microRNAin combination are sufficient to form an invasive cleavage structure. 7.The method of claim 5, further comprising a second nucleic acid, whereinsaid second nucleic acid contains a first region that is complementaryto a portion of said microRNA target that is not complementary to saidfirst nucleic acid, and a second region that is not complementary tosaid microRNA target, wherein said second region of said second nucleicacid forms a hairpin loop to form a half dumbbell structure when saidnucleic acid is hybridized to said microRNA.
 8. The method of claim 1,wherein said detecting comprises detecting cleavage of an invasivecleavage structure.
 9. The method of claim 1, wherein said samplecomprises a cell lysate.
 10. The method of claim 1, wherein a pluralityof different microRNAs are detected.
 11. The method of claim 10, whereinsaid plurality of microRNAs comprise a first microRNA and a secondmicroRNA that is said first microRNA having a polymorphism.
 12. Themethod of claim 1, further comprising detecting a second nucleic acidtarget.
 13. The method of claim 12, wherein said second nucleic acidtarget is RNA.
 14. The method of claim 13, wherein said second nucleicacid target is selected from the group consisting of U6 and GAPDH. 15.The method of claim 1, wherein said microRNA is selected from the groupconsisting of Let-7, miR-1, miR-135, miR-15, miR-16, miR125b, miR-1d,and miR124a.